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Pasteurella multocida is an important cause of pneumonic pasteurellosis in small ruminants. Its prevalence was investigated in 349 pneumonic lungs from sheep (n = 197) and goats (n = 152), and genotypes of isolates were determined by capsular and lipopolysaccharide (LPS) typing as well as by virulotyping based on the detection of 12 virulence-associated genes. P. multocida was isolated from 29.4 % of sheep lungs and 13.8 % of goat lungs. A (78.5 %) and D (21.5 %) capsular types, as well as L3 (41.8 %) and L6 (57.0 %) LPS genotypes, were detected, with the A:L6 genotype being the most prevalent in both sheep (59.6 %) and goat (52.4 %) isolates. A total of 19 virulence profiles (VP) were detected, seven non-toxigenic and 12 toxigenic, which correlated with the capsular-LPS genotype. All isolates of each VP belonged to the same LPS and capsular genotype, except for one isolate of VP1. The diversity in VP was higher among toxigenic (0.29) than non-toxigenic (0.18) isolates. Moreover, the toxigenic VPs showed more diversity in their capsular-LPS genotypes, with the two main toxigenic VPs belonging to genotypes D:L3 (VP2) and A:L3 (VP3). Therefore, the abundance of toxigenic isolates among sheep and goat isolates does not seem to correspond to the expansion of a more virulent lineage associated with pneumonic pasteurellosis in small ruminants. The most prevalent genotypes among sheep isolates were the non-toxigenic VP1:A:L6 (41.4 %) and the toxigenic VP3:A:L3 (17.2 %) genotypes, whereas the most prevalent among goat isolates were the toxigenic VP2:D:L3 (33.3 %) and the non-toxigenic VP1:A:L6 (14.3 %) and VP4:A:L6 (14.3 %) genotypes. These prevalent toxigenic and non-toxigenic genotypes seem to be epidemiologically relevant in pneumonic pasteurellosis of small ruminants., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Bacterial pathogens and commensals are surrounded by diverse surface polysaccharides which include capsules and lipopolysaccharides. These carbohydrates play a vital role in bacterial ecology and interactions with the environment. Here, we review recent rapid advancements in this field, which have improved our understanding of the roles, structures, and genetics of bacterial polysaccharide antigens. Genetic loci encoding the biosynthesis of these antigens may have evolved as bacterial diversity-generating machines, driven by selection from a variety of forces, including host immunity, bacteriophages, and cell-cell interactions. We argue that the high adaptive potential of polysaccharide antigens should be taken into account in the design of polysaccharide-targeting medical interventions like conjugate vaccines and phage-based therapies., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Xanthomonas translucens pv. translucens (Xtt) is a Gram-negative pathogen of crops from the plant family Poaceae. The lipopolysaccharide (LPS) of Xtt was isolated and chemically characterized. The analyses revealed the presence of rhamnose, xylose, mannose, glucose, galacturonic acid, phosphates, 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo) and fatty acids (10:0, 11:0, 11:0(3-OH) i/a, 11:0(3-OH), 12:0(3-OH) i/a, 12:0(3-OH), 12:0, 13:0(3-OH) i, 13:0(3-OH) a, 13:0(3-OH), 14:0(3-OH) i/a, 14:0(3-OH) and 16:0). The rough type of LPS (lipooligosaccharides; LOS) was isolated and its composition determined utilizing mass spectrometry. The structure of core-lipid A backbone was revealed by nuclear magnetic resonance (NMR) spectroscopy performed on O-deacylated LOS sample, and was shown to be: α-D-Manp-(1→3)-α-D-Manp-(1→3)-β-D-Glcp-(1→4)-α-D-Manp-(1→5)-α-Kdo-(2→6)-β-D-GlcpN-(1→6)-α-D-GlcpN. 4-α-Man and Kdo were further substituted via phosphodiester groups by two galactopyranuronic acids. Xtt LPS elicited a stress response in Nicotiana tabacum suspension cell cultures, namely a transient calcium signal and the generation of H2O2 was observed. Pharmacological studies indicated the involvement of plasma membrane calcium channels, kinases and phospholipase C as key factors in Xtt LPS induced pathogen signaling.

Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog ( Ceratophyrus ornate ) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi . We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata -like strain BO2, with traits that depart significantly from those of the "classical" Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella , but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.

The aim of this study was to compare different typing methods, individually and combined, for use in the monitoring of Campylobacter in food. Campylobacter jejuni (n=94) and Campylobacter coli (n=52) isolated from different broiler meat carcasses were characterized using multilocus sequence typing (MLST), flagellin gene A restriction fragment length polymorphism typing (flaA-RFLP), antimicrobial resistance profiling (AMRp), the presence/absence of 5 putative virulence genes; and, exclusively for C. jejuni, the determination of lipooligosaccharide (LOS) class. Discriminatory power was calculated by the Simpson's index of diversity (SID) and the congruence was measured by the adjusted Rand index and adjusted Wallace coefficient. MLST was individually the most discriminative typing method for both C. jejuni (SID=0.981) and C. coli (SID=0.957). The most discriminative combination with a SID of 0.992 for both C. jejuni and C. coli was obtained by combining MLST with flaA-RFLP. The combination of MLST with flaA-RFLP is an easy and feasible typing method for short-term monitoring of Campylobacter in broiler meat carcass., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Salmonella enterica subsp. enterica serovar (serotype) Abortusovis is a member of the Enterobacteriaceae. This serotype is naturally restricted to ovine species and does not infect humans. Limited information is available about the immune response of sheep to S. Abortusovis. S. Abortusovis, like Salmonella enterica subsp. enterica serovar Typhi, causes a systemic infection in which, under natural conditions, animals are not able to raise a rapid immune response. Failure to induce the appropriate response allows pathogens to reach the placenta and results in an abortion. Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns (PAMPs) that are specific to bacteria and are not synthesized by the host. Toll-like receptors (TLRs) are a family of receptors that specifically recognize PAMPs. As a first step, we were able to identify the presence of Toll-like receptor 4 (TLR4) on the ovine placenta by using an immunohistochemistry technique. To our knowledge, this is the first work describing the interaction between S. Abortusovis LPS and TLR4. Experiments using an embryonic cell line (HEK293) transfected with human and ovine TLR4s showed a reduction of interleukin 8 (IL-8) production by S. Abortusovis and Salmonella enterica subsp. enterica serovar Paratyphi upon LPS stimulation compared to Salmonella enterica subsp. enterica serovar Typhimurium. Identical results were observed using heat-killed bacteria instead of LPS. Based on data obtained with TLR4 in vitro stimulation, we demonstrated that the serotype S. Abortusovis is able to successfully evade the immune system whereas S. Typhimurium and other serovars fail to do so., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Lipoteichoic acids (LTA) are polymers of alternating units of a polyhydroxy alkane, including glycerol and ribitol, and phosphoric acid, joined to form phosphodiester units that are found in the envelope of Gram-positive bacteria. Here we review four different types of LTA that can be distinguished on the basis of their chemical structure and describe recent advances in the biosynthesis pathway for type I LTA, d-alanylated polyglycerol-phosphate linked to di-glucosyl-diacylglycerol. The physiological functions of type I LTA are discussed in the context of inhibitors that block their synthesis and of mutants with discrete synthesis defects. Research on LTA structure and function represents a large frontier that has been investigated in only few Gram-positive bacteria.

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.

The lipopolysaccharide is the major component of Gram-negative bacteria outer membrane. LPS comprises three covalently linked regions: the lipid A, the rough core oligosaccharide, and the O-antigenic side chain determining serotype specificity. Wild-type LPS (sLPS) contains the O-antigenic side chain and is referred to as smooth. Rough LPS (rLPS) does not contain the O-side chain. Most wt bacteria and especially wt Enterobacteriaceae express prevalently the sLPS form although some truncated rLPS molecules always reach the external membrane. The two sLPS and rLPS forms are used almost indistinctly to study the effects on innate immune cells. Nevertheless, there is evidence that their mechanism of action may be different. For instance, while sLPS requires CD14 for the initiation of both MyD88-dependent and independent signal transduction pathways at least at low doses, rLPS leads to MyD88-dependent responses in the absence of CD14 even at low doses. Here we have identified additional differences in the signaling capacity of the two LPS species in the mouse. We have found that rLPS, diversely from sLPS, is capable of activating in dendritic cells (DCs) the Ca(2+)/calcineurin and NFAT pathway in a CD14-independent manner, moreover it is also capable per se of activating the inflammasome and eliciting IL-1β secretion independent of the presence of additional stimuli required instead for sLPS. The ability of rLPS of activating the inflammasome in vitro has as a direct consequence a higher efficiency of rLPS-exposed DCs in activating natural killer (NK) cells compared to sLPS-exposed DCs. However, diversely from possible predictions, we found that the different efficiencies of the two LPS species in eliciting innate responses are almost nullified in vivo. Therefore, sLPS and rLPS induce nearly similar in vivo innate responses but with different mechanisms of signaling., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aim: Subtyping of lipooligosaccharides (LOS) of non-typeable strains of Haemophilus influenzae (NTHi) isolated from children with bronchopulmonary diseases., Materials and Methods: Lipooligosaccharides obtained from 62 acapsular strains of H. influenzae were studied by vertical SDS-electrophoresis in PAAG., Results: Majority of LOS formed electrophoretically mobile components in low molecular mass zone. Obtained results allowed to differentiate 23 subtypes of LOS. Lipooligosaccharides of majority of strains (67.7%) belonged to one of 10 main subtypes, 30.6% of strains belonged to mixed subtypes because they had signs of 2-3 subtypes., Conclusion: Strains possessing LOS of three subtypes--VI, VII, and X--were significantly more prevalent in pediatric patients (p < 0.05). More than one third (43.5%) of studied NTHi strains belonged to these subtypes.

Aggregatibacter actinomycetemcomitans is divided into 6 serotypes. Occurrence of non-serotypeable strains is known, but background reasons are unclear. We hypothesized that non-serotypeable strains represent new serotypes or have altered expression of serotype-specific polysaccharide antigen (S-PA). We first characterized 311 strains from 189 individuals using both immunoassay- and PCR-based serotyping. Next, using natural human infection and rabbit immunization approaches, we clarified whether the phenotypically non-serotypeable strains expressed S-PA. Immunoassay identified serotypes a-f among 216 strains from 159 individuals. The remaining 95 strains from 30 individuals were phenotypically non-serotypeable. Yet, all these strains were identified by PCR-typing as serotype a-, b-, c-, or f. Non-serotypeability was confirmed by Western immunoblot with respective rabbit antisera. Patient sera remained non-reactive with autologous non-serotypeable strains at the serotype-specific region. Rabbit immunization with a phenotypically non-serotypeable strain induced no antibody production against S-PA. Thus, phenotypically non-serotypeable strains did not include novel serotypes, but lacked S-PA expression.

The genus Burkholderia includes many bacteria that cause serious human infections. As is the case with other Gram-negative bacteria, Burkholderia species produce LPS, which is an abundant component of the bacterial cell surface. Burkholderia cepacia complex (Bcc) bacteria (which include at least 17 separate species) produce LPS structures that are quite different. In an attempt to determine the degree of LPS epitope variation among Bcc species, a mAb was produced, designated 5D8, specific for the LPS of B. cepacia. Western blot analysis determined that mAb 5D8 was able to produce the classic 'ladder pattern' when used to probe B. cepacia and Burkholderia anthina lysates, although 5D8 did not produce this pattern with the other seven Bcc species tested. mAb 5D8 reacted with varying intensity to most but not all of the additional B. cepacia and B. anthina strains tested. Therefore, there seems to be significant epitope variation among Bcc LPS both between and within species. Additionally, mAb 5D8 reacted with a proteinase-K-sensitive 22 kDa antigen in all Bcc strains and also in a strain of Burkholderia pseudomallei.

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.

The O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide of Citrobacter werkmanii PCM 1548 and PCM 1549 (serogroup O14) and found to contain D-glucose, D-glucosamine and glycerol-1-phosphate in molar ratios 2 : 2 : 1. Based on methylation analysis and 1H and 13C nuclear magnetic resonance spectroscopy data, it was established that the O-specific polysaccharides from both strains have the identical branched tetrasaccharide repeating unit with 3,6-disubstituted GlcNAc, followed by 2,4-disubstituted Glc residues carrying at the branching points lateral residues of Glc and GlcNAc at positions 6 and 2, respectively. Glycerol-1-phosphate is linked to position 6 of the chain Glc. All sugars have a beta configuration, except for the side-chain Glc, which is alpha. Serological studies revealed a close relatedness of the lipopolysaccharides of C. werkmanii PCM 1548 and PCM 1549, both belonging to serogroup O14. In immunoblotting, anti-C. werkmanii PCM 1548 serum showed no cross-reactivity with the O-polysaccharide bands of the lipopolysaccharides of Citrobacter youngae PCM 1550 (serogroup O16) and Hafnia alvei PCM 1207, also containing a lateral glycerol phosphate residue.

A fast microchip electrophoresis method was developed to analyze and differentiate bacterial endotoxins directly from whole-cell lysates after removal of the proteinaceous components with proteinase K digestion and a precipitation of the endotoxin components. The partially purified endotoxin components were visualized by the interaction with dodecyl sulphate and then a fluorescent dye. The lipopolysaccharide (LPS) profiles can be directly evaluated from digested bacterial cells, and the electrophoresis patterns very closely resembled to those of pure LPSs, and the R and S chemotypes can be used to assign the strains. The method has been found to be useful in the screening of a large number of bacterial mutants and the structural characterization of endotoxins extracted only from 1 ml cultures.

In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14DeltaaroA, was constructed. PA14DeltaaroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14DeltaaroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14DeltaaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa DeltaaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14DeltaaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1DeltaaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14DeltaaroA vaccine's protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.

Background: Porphyromonas gingivalis is a Gram-negative bacterium that is an important etiologic agent of human adult periodontitis. The goal of the study was to test the hypothesis that two isoforms of P. gingivalis lipopolysaccharide (PgLPS), PgLPS(1435)(/1449) and PgLPS(1690), exhibit differences in their capacity to stimulate systemic versus local responses compared to Escherichia coli lipopolysaccharide (LPS)., Methods: LPS was inoculated into the scalp of mice, and the response was measured locally at the site of inoculation and systemically in the heart/aorta. Vascular cell adhesion molecule (VCAM)-1 was assessed at the protein level by enzyme-linked immunosorbent assay, and VCAM-1, E-selectin, and intercellular adhesion molecule (ICAM)-1 were assessed at the RNA level of the RNase protection assay. Serum tumor necrosis factor-alpha (TNF-alpha) levels were also measured., Results: E. coli LPS and both isoforms of P. gingivalis LPS were relatively potent in stimulating the expression of inflammatory markers, with E. coli LPS being more potent. In contrast, when the systemic response was measured in the heart/aorta, E. coli LPS, but not P. gingivalis LPS, significantly induced inflammatory markers. At moderate to low doses (1 and 10 microg per injection), serum TNF-alpha levels were minimally induced by P. gingivalis LPS compared to E. coli LPS., Conclusions: Both forms of P. gingivalis LPS stimulated an inflammatory response when injected into connective tissue but were minimally stimulatory when a systemic response was measured. In contrast, E. coli LPS was a potent stimulus at the systemic and local levels.

A lipopolysaccharide (LPS) and beta-1,3-glucan binding protein (LGBP) gene was cloned from hemocytes of kuruma shrimp Marsupenaeus japonicus by reverse-transcription polymerase chain reaction (RT-PCR), cloning and sequencing of overlapping PCR, and rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of M. japonicus LGBP is 1062 bp and encodes a 354 amino acid (aa) sequence with a 23 aa signal peptide. The calculated molecular mass of the mature protein (331 aa) is 40.15 kDa with an estimated pI of 4.78. The M. japonicus LGBP sequence contains (1) two putative N-linked glycosylation sites, (2) two putative integrin-binding motifs, (3) a kinase C phosphorylation site (KCPS), (4) a glucanase motif (GM), and (5) two potential polysaccharide recognition motifs (polysaccharide binding motif (PsBM) and beta-glucan recognition motif (GRM)), and with features of tryptophan-rich, slight homology to lysozyme, and slight homology to lectin. A sequence comparison showed that the deduced amino acids of M. japonicus LGBP has an overall high similarity to penaeid LGBP and betaGBP (85.6-89.9%), lobster Homarus gammarus betaGBP (77.0%), and crayfish Pacifastacius leniusculus LGBP (67.8%). The phylogenetic analysis revealed that M. japonicus LGBP grouped together with other crustacean LGBP and betaGBP, and was close to termite GNBP, but was far way from moth betaGBP, betaGRP, fly GNBP, and mosquito betaGRP. The LGBP of M. japonicus was strongly expressed in hemocytes. The LGBP mRNA transcript in hemocytes of M. japonicus was significantly upregulated 12-48 h after a LPS injection, indicating activation of the innate immune system through the binding of the LGBP and LPS complex.

Heterogeneous patterns were obtained for lipopolysaccharide (LPS) from 1,327 Burkholderia pseudomallei isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining, and immunoblot analysis. Two LPS serotypes (A and B) possessing different ladder profiles and a rough LPS without ladder appearances were identified. All three LPS types were antigenically distinct by immunoblotting. The predominant type A (97%) produced the lowest amount of biofilm. The two less common types (smooth type B and rough type) were found more in clinical than environmental isolates and more in Australian isolates than Thai isolates. These isolates were more often associated with relapse than with primary infection.

More than 90% of all publications on endotoxin were carried out with endotoxins (lipopolysaccharide, LPS) from enterobacteriaceae. We compared the immune stimulatory potency of 11 different LPSs using human whole blood incubations. While the majority of LPSs induced cytokine release equipotently, a 1,000-fold more LPS from Pseudomonas aeruginosa or Vibrio cholerae was still less potent in inducing TNF, IL-1 beta, IL-10 and IFN-gamma though it potently induced nanogram quantities IL-8. All LPSs tested, regardless of the micro-organism, showed Toll-like receptor (TLR)4-dependence, except for the LPSs from P. aeruginosa and V. cholerae, which were both TLR4- and TLR2-dependent. Interestingly, UV-inactivated P. aeruginosa bacteria, although Gram-negative, also showed TLR2- and TLR4-dependence. Re-purification of commercial LPS preparations by phenol re-extraction led to a complete loss of the TLR2 dependency, indicating contamination with lipoproteins. In the Limulus amebocyte lysate assay, often performed to exclude contamination in purified water likely to originate from P. aeruginosa, P. aeruginosa LPS was only 2-fold less potent than LPS from S. abortus equi or the assay standard LPS from E. coli. This results in an overestimation of pyrogenic burden by a factor of 500 in the sample when compared with the biological activity of highly purified P. aeruginosa LPS in human whole blood.

Introduction: Gram-negative bacteria of genus Proteus are common human intestinal and urinary tract pathogens. In the genus Proteus there are four clinically important named species: P. mirabilis, P. vulgaris, P. penneri, and P. hauseri, and three unnamed Proteus genomospecies: 4, 5, and 6. The clinical significance of P. penneri, described in 1982 as a new species, is poorly documented. The aim of this work is serological characterization and classification of a ceftriaxone-susceptible P. penneri S29 strain isolated from a 34-year-old patient with postneurosurgical meningitis. In this characterization we will also include a ceftriaxonresistant strain, P. penneri R15, isolated from the same patient after 12 days' treatment with ceftriaxon and other antibiotics., Material/methods: Rabbit polyclonal O-antisera were obtained against these two strains and purified and lipopolysaccharides (LPS) were extracted from the bacterial mass of the P. penneri S29 and R15 strains. In the serological investigations the following tests were used: enzyme immunosorbent assay (EIA), passive immunohemolysis (PIH), inhibition of these tests, absorption of rabbit O-antisera with the respective LPS, and repeated PIH, SDS/PAGE, and Western blot techniques., Results: The serological studies of the LPS extracted from both P. penneri strains showed the identity of both preparations of O-polysaccharides from LPS. In P. penneri S29 O-antiserum, four different types of antibodies were described and characterized., Conclusions: Both investigated P. penneri S29 and R15 strains were classified to the Proteus O31ab serogroup.

Lipopolysaccharides (LPS), otherwise termed 'endotoxins', are outer-membrane constituents of Gram-negative bacteria. Lipopolysaccharides play a key role in the pathogenesis of 'Septic Shock', a major cause of mortality in the critically ill patient. Therapeutic options aimed at limiting downstream systemic inflammatory processes by targeting lipopolysaccharide do not exist at the present time. We have defined the pharmacophore necessary for small molecules to specifically bind and neutralize LPS and, using animal models of sepsis, have shown that the sequestration of circulatory LPS by small molecules is a therapeutically viable strategy. In this paper, the interactions of a focused library of lysine-spermine conjugates with lipopolysaccharide (LPS) have been characterized. Lysine-spermine conjugates with the epsilon-amino terminus of the lysinyl moiety derivatized with long-chain aliphatic hydrophobic substituents in acyl or alkyl linkage bind and neutralize bacterial lipopolysaccharides, and may be of use in the prevention or treatment of endotoxic shock states.

Host responses during pneumonic pasteurellosis were compared in sheep infected with strains of Mannheimia (Pasteurella) haemolytica A1 differing in their O-antigen type. Nine-week-old, specific pathogen-free lambs were infected intratracheally with parainfluenza type 3 virus (10(8) TCID(50)) followed 7 days later by 5-6 x 10(7) CFU of M. haemolytica A1 possessing rough (group R, 6 lambs) or smooth (group S, 6 lambs) lipopolysaccharide, or saline (group C, 4 lambs). Group C lambs remained afebrile with no evidence of endotoxaemia or bacteraemia and biochemical parameters were normal. Group R and group S lambs became febrile within 2-3 h postinfection and the response was higher and more prolonged in group R lambs. Four group R and 2 group S lambs developed clinical pneumonic pasteurellosis within 24-48 h and the severity of disease correlated with episodes of endotoxaemia, bacteraemia and elevated eicosanoid concentrations. At post-mortem, M. haemolytica (10(7)-10(9) CFU/g) was isolated from the lungs of all 6 group R lambs but from only 1 group S lamb. The results indicate an association between the incidence and severity of ovine pneumonic pasteurellosis and LPS chemotype and suggest an important role for LPS chemotype in determining host-species susceptibility to lung infection.

Surfactant protein D (SP-D) plays important roles in the regulation of innate immune responses in the lung. We have previously shown that SP-D can agglutinate and enhance the macrophage-dependent killing of specific unencapsulated phase variants of Klebsiella pneumoniae. In the present studies, we used 16 clinical isolates of Klebsiella representing four O-serotypes and examined the interaction of SP-D with their isolated LPSs. Although SP-D bound to the core oligosaccharide of rough LPS from all isolates, it selectively bound to smooth forms of LPS expressed by O-serotypes with mannose-rich repeating units in their O-polysaccharides. SP-D was more potent in agglutinating unencapsulated phase variants of O-serotypes expressing these SP-D "reactive" O-polysaccharides, and more effectively inhibited the adhesion of these serotypes to lung epithelial cells. This novel anti-adhesion activity required the multimerization of trimeric SP-D subunits (dodecamers). Klebsiella serotypes expressing "nonreactive" LPS O-Ags were isolated at a significantly higher frequency from patients with K. pneumoniae. Our findings suggest that SP-D plays important roles in the clearance of opportunistic Gram-negative bacteria and contributes to known serotypic differences in the pathogenicity of Klebsiella through specific interactions with O-polysaccharides.

Mucosal immunity in the gastrointestinal (GI) tract is a primary defence against GI pathogens. We hypothesise that a mucosal response to lipopolysaccharide (LPS), especially to the common (core) determinants of GI pathogenic Escherichia coli strains, is protective. The aims of this study were to investigate the specificities, levels and development of humoral responses in health and GI disease to the R3 LPS core and O-polysaccharide of E. coli O157. The purpose was to try to predict whether vaccination or passive immunisation might induce protection. Wherever possible, paired whole gut lavage fluid (WGLF) and serum samples were collected for comparison of the mucosal and systemic responses. Matched saliva samples were also collected from some study groups. The patient groups included those with acute E. coli O157 disease (serum only), patients convalescing after E. coli O157 infections, and patients undergoing routine investigation for GI conditions but subsequently shown to be immunologically normal. Some samples of WGLF from patients with Crohn's disease (CRO) and ulcerative colitis (UC) were included to allow comparisons with patients with inflammatory conditions known to alter antibody secretion in the GI tract. The healthy groups from whom serum and saliva only were taken included blood donors, healthy volunteers and a group of slaughterhouse workers. This latter group was likely to have been exposed regularly to faecal bacteria from animals and antibody specificities might have been expected to be different from other healthy individuals. Levels and classes of antibodies were determined by ELISA with microtitration plates coated with polymyxin complexes of whole LPS extracted from E. coli O157 and LPS from the E. coli R3 rough mutant. Antibodies of IgG and IgM classes were measured in serum and IgA was measured in WGLF and saliva. IgG antibodies to the O157 LPS and the R3 core oligosaccharide were detected in the serum of healthy blood donors. Patients with acute E. coli O157 disease showed elevated levels of serum IgM to O157 LPS and R3, with IgG levels raised only to R3. In serum from convalescent patients, IgG to O157 LPS was significantly above the control groups only in the period 6-16 weeks after infection. Total IgA levels were similar in WGLF specimens from all groups, except the patients with UC, whose levels were much higher. Specific IgA levels were higher in the E. coli O157 convalescent group, but there were no significant correlations overall. UC patients had significantly lower levels of IgA to O157 and CRO patients had higher O157 IgA levels than UC patients and healthy volunteers. In serum, inhibition of ELISA showed that the response to the O157 LPS was due in part to a response to the R3 oligosaccharide component. This response was much more pronounced in the healthy and non-O157 groups than in convalescent patients. There was no correlation between specific IgA antibody levels in saliva and matched specimens of WGLF, and levels in sequential saliva specimens fluctuated widely. The significant IgG and IgA responses to the R3 core suggest that there is immunological memory to this oligosaccharide LPS component which may have a role in protection against E. coli LPS both systemically and locally in the GI tract. Boosting of this mucosal response to the LPS core, either naturally through exposure or by active or passive immunisation, may confer protection. Finally, antibody responses to E. coli O157 must be interpreted with caution, as the response detected is a sum of responses to the O-specific polysaccharide and the R3 core.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigens Lewis x (Le(x)), Le(y), and H type I. In this report, we demonstrate that the H type I epitope displays high-frequency phase variation. One variant expressed Le(x) and Le(y) and no H type I as determined by serology; this switch was reversible. Insertional mutagenesis in NCTC 11637 of JHP563 (a poly(C) tract containing an open reading frame homologous to glycosyltransferases) yielded a transformant with a serotype similar to the phase variant. Structural analysis of the NCTC 11637 LPS confirmed the loss of the H type I epitope. Sequencing of JHP563 in strains NCTC 11637, an H type I-negative variant, and an H type I-positive switchback variant showed a C14 (gene on), C13 (gene off), and C14 tract, respectively. Inactivation of strain G27, which expresses Le(x), Le(y), H type I, and Le(a), yielded a transformant that expressed Le(x) and Le(y). We conclude that JHP563 encodes a beta3-galactosyltransferase involved in the biosynthesis of H type I and Le(a) and that phase variation in H type I is due to C-tract changes in this gene. A second H type I-negative variant (variant 3a) expressed Le(x) and Le(a) and had lost both H type I and Le(y) expression. Inactivation of HP093-HP094 resulted in a transformant expressing Le(x) and lacking Le(y) and H type I. Structural analysis of a mutant LPS confirmed the serological data. We conclude that the HP093-HP094 alpha2-fucosyltransferase (alpha2-FucT) gene product is involved in the biosynthesis of both Le(y) and Le(x). Finally, we inactivated HP0379 in strain 3a. The transformant had lost both Le(x) and Le(a) expression, which demonstrates that the HP0379 gene product is both an alpha3- and an alpha4-FucT. Our data provide understanding at the molecular level of how H. pylori is able to diversify in the host, a requirement likely essential for successful colonization and transmission.

Campylobacter jejuni, a Gram-negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post-infection neurological complications of the Miller-Fisher and Guillain-Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one-third of strains also producing high-molecular-weight LPS (referred to as O-antigen). In this report, we demonstrate the presence of the high-molecular-weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O-antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O-antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.

This study describes the molecular makeup of the cell-wall lipopolysaccharides (LPSs) (O-chain polysaccharide-->core oligosaccharide-->lipid A) from five Helicobacter pylori strains: H. pylori 26695 and J99, the complete genome sequences of which have been published, the established mouse model Sydney strain (SS1), and the symptomatic strains P466 and UA915. All chemical and serological experiments were performed on the intact LPSs. H. pylori 26695 and SS1 possessed either a low-Mr semi-rough-form LPS carrying mostly a single Ley type-2 blood-group determinant in the O-chain region covalently attached to the core oligosaccharide or a high-Mr smooth-form LPS, as did strain J99, with an elongated partially fucosylated type-2 N-acetyllactosamine (polyLacNAc) O-chain polymer, terminated mainly by a Lex blood-group determinant, connected to the core oligosaccharide. In the midst of semi-rough-form LPS glycoforms, H. pylori 26695 and SS1 also expressed in the O-chain region a difucosylated antigen, alpha-L-Fucp(1-3)-alpha-L-Fucp(1-4)-beta-D-GlcpNAc, and the cancer-cell-related type-1 or type-2 linear B-blood-group antigen, alpha-D-Galp(1-3)-beta-D-Galp(1-3 or 4)-beta-D-GlcpNAc. The LPS of H. pylori strain P466 carried the cancer-associated type-2 sialyl Lex blood-group antigen, and the LPS from strain UA915 expressed a type-1 Leb blood-group unit. These findings should aid investigations that focus on identifying and characterizing genes responsible for LPS biosynthesis in genomic strains 26695 and J99, and in understanding the role of H. pylori LPS in animal model studies. The LPSs from the H. pylori strains studied to date were grouped into specific glycotype families.

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a protective and permeability barrier. X-ray diffraction analysis of osmotically stressed LPS multilayers was used to determine the structure and interactive properties of LPSs from strains containing the minimum number of sugars necessary for bacterial survival (Re chemotype) to the maximum number of sugars found in rough bacteria (Ra chemotype). At 20 degrees C in the absence of divalent cations, LPS suspensions gave a sharp wide-angle reflection at 4.23 A and a broad low-angle band centered at 50-68 A depending on the chemotype, indicating the presence of gel phase bilayers separated by large fluid spaces. As osmotic pressure was applied, the apposing bilayers were squeezed together and lamellar diffraction at 6 A resolution was obtained. At low applied pressures (<10(6) dyn/cm2), the total repulsive pressure between bilayers could be explained by electrostatic double layer theory. At higher applied pressures, there was a sharp upward break in each pressure-distance relation, indicating the presence of a hydrophilic steric barrier whose range depended strongly on the LPS chemotype. The positions of these upward breaks, along with electron density profiles, showed that the sugar core width systematically increased from 10 A for the Re chemotype to 27 A for the Ra chemotype. In excess buffer, the addition of divalent cations brought the bilayers into steric contact. Electron density profiles were used to determine the locations of cation binding sites and polar substituents on the LPS oligosaccharide core. The area per hydrocarbon chain was approximately 26 A2 in liquid-crystalline LPS bilayers, an indication of an acyl chain packing that is much tighter than that found in bilayers composed of typical membrane lipids. This unusually tight packing could be a critical factor in the permeability barrier provided by LPS.

Background: The Gram-negative facultatively anaerobic coccobacillus Actinobacillus actinomycetemcomitans is the major pathogen in localized juvenile periodontitis (LJP) and some forms of adult periodontitis (AP). A. actinomycetemcomitans can be grouped into 5 serotypes (a through e) based on differences in the carbohydrate moiety of cell surface lipopolysaccharide. The A. actinomycetemcomitans population is genetically heterogeneous. Since the studies on A. actinomycetemcomitans colonization have mostly applied only culture techniques, the clonality of the follow-up isolates has not been established. Thus, it is possible that, although A. actinomycetemcomitans could be repeatedly isolated from an individual, the initial colonizing strain was replaced by another strain. The aim of the study was to determine whether oral A. actinomycetemcomitans strains change spontaneously over time or after periodontal treatment., Methods: A total of 922 A. actinomycetemcomitans isolates were recovered from 115 subjects. From each subject A. actinomycetemcomitans isolates were obtained from 2 to 9 follow-up samples 0.5 to 11.5 years apart. After the first sampling occasion, 99 subjects were treated for either LJP or AP, whereas the 16 non-periodontitis subjects received no treatment. All A. actinomycetemcomitans isolates were serotyped and 235 isolates from 52 subjects genotyped with AP-PCR and/or with ribotyping., Results: Isolates of only one serotype, or non-serotypeable isolates alone, were repeatedly found in 104 subjects; serotype a occurred in 25%, b in 33%, c in 23%, d in 5%, e in 7%, and non-serotypeable isolates in 8% of these subjects. Two serotypes (or serotypeable isolates together with non-serotypeable isolates) occurred simultaneously in 9 subjects and in each of these subjects at least one of the serotypes was detected at each sampling occasion. In one subject the initial serotype reappeared although a different serotype was once seen alone, whereas in another subject the initial serotype could not be recovered later. Identical genotypes of A. actinomycetemcomitans were repeatedly detected in each of 52 subjects with follow-up isolates of the same serotype., Conclusions: The results showed that spontaneous or treatment-induced change in the oral A. actinomycetemcomitans strain(s) is extremely rare and that colonization with the same strain(s) seems to be remarkably persistent.

We have previously described an inhibition enzyme-linked immunosorbent assay method for the O typing of O1 lipopolysaccharide from Klebsiella pneumoniae which overcomes the technical problems and limitations of the classical O-typing method. In this study, we have extended the method to all of the currently recognized O types. The method was validated by studying the prototype strains that have defined the O groups by the classical tube agglutinatination O-typing method. Based on these results, we confirmed the O types of 60 of 64 typeable strains, and we propose a revised O-antigenic scheme, with minor but necessary changes, consisting of serogroups or serotypes O1, O2, O2ac, O3, O4, O5, O7, O8, and O12. Application of this typing method to 638 K. pneumoniae clinical isolates from Denmark, Spain, and the United States from different sources (blood, urine, and others) showed that up to 80% of these isolates belong to serotypes or serogroups O1, O2, O3, and O5, independently of the source of isolation, and that a major group of nontypeable isolates, representing about 17% of the total, consists of half O+ and half O- strains. Differences were observed, however, in the prevalence of the lipopolysaccharide O types or groups, depending on the country and isolation source.

We previously reported the case of a human chronic Bordetella bronchiseptica respiratory infection, due to contact with infected rabbits. Lipopolysaccharides of the human isolates, of one rabbit isolate and of isolate from other origins were analyzed with sera from infected mice, rabbit and human. Antigenicity and length of the lipopolysaccharide molecules varied between isolates. We showed a progressive loss of O-chain during infection, associated with an enhanced susceptibility of the isolates to the bactericidal effect of normal serum. This observation suggests the existence of an intracellular niche which selects for strains with distinct lipopolysaccharide types.

The lipopolysaccharides LPS I and LPS II, isolated from the hypovirulent EV40 strain of Yersinia pestis, are composed only of type R lipopolysaccharides. This type consists of two forms a and b, depending on their solubility pattern in a solvent mixture containing varying proportions of chloroform, methanol, hexane, and hydrochloric acid. LPS I consists of one subtype, RIb, while LPS II consists of two subtypes, RIIa and RIIb. Analysis by gel electrophoresis shows that the mass of these lipopolysaccharide forms are in the vicinity of 2000-3000 Da. The RIb and RIIb subtypes, which are found in the majority of lipopolysaccharide I and II fractions, are composed of ketoses and amines that are similar to those occurring in LPS I and LPS II. In contrast, the two subtypes RIIa and RIIb are different both in terms of the composition of lipid A and the extent of its substitution. Certain fractions of RIIa contain only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), while other fractions of RIIb possess a lipid A, which is not substituted by arabinose. The whole set of these R-type lipopolysaccharide forms are excellent models for the study of the role of the primary structure of the polysaccharide region, and for the effect of lipid A substitution on the biological activity of bacterial lipopolysaccharides.

Nitric oxide (NO) plays a complex role in the modulation of the inflammatory response, having either a pro-inflammatory or a protective role. Actinobacillus actinomycetemcomitans is considered an important etiological agent in localized juvenile periodontitis. We have studied the effect of lipopolysaccharide (LPS) extracted from this periodontopathogenic bacterium on NO synthesis in an in vitro murine macrophage system. LPS from A. actinomycetemcomitans induced a significant production of NO even at concentrations as low as 1 ng/ml, whereas LPS from E. coli had to be added in concentrations of 100 ng/ml to obtain similar effects. Production of NO was blocked by NG-nitro-L-arginine methylester, and pre-treatment of LPS from A. actinomycetemcomitans with polymyxin B abolished the production of NO, while prostaglandin E2 enhanced the synthesis of NO.

Lipopolysaccharides (LPS) of Rhizobium galegae, a symbiotically nitrogen-fixing species of root-nodule bacteria, were isolated by the phenol-water method from strain HAMBI 1461, the LPS of which resembled enterobacterial smooth type LPS, and from strains HAMBI 1174 and HAMBI 1208, the LPSs of which resembled rough type LPS. The results of PAGE analysis of LPSs, Bio-Gel P2 gel filtration of polysaccharide fractions and the presence of deoxysugars and 4-O-methyl-deoxysugar both in the rough and smooth LPSs suggested that rough LPS contained a short O-antigenic polysaccharide for which we propose the name short O-chain LPS. Accordingly, the smooth LPS is called long O-chain LPS. Despite of the differences in the structure of LPS of R. galegae, all strains were equally effective in nodulating their hosts. The short O-chain LPS of R. galegae showed many features similar to those of phylogenetically related agrobacteria.

Rhodococcus rhodnii N445 was investigated for the presence of macroamphiphilic lipoglycan. Purification of a hot phenol-water extract by hydrophobic interaction chromatography allowed the resolution of three lipoglycan fractions. The two main preparations contained lipoglycans with carbohydrate compositions consistent with the presence of lipoarabinomannan and lipomannan, whilst the minor fraction appeared to contain a mixture of these two lipoglycans. The fatty acid composition of the lipoglycans resembled that of the whole cells except that the relative proportion of unsaturated fatty acids was decreased. Although lipoarabinomannan and structurally-related lipomannan lipoglycans from representatives of the genus Mycobacterium have been extensively studied, this is the first report of the lipoglycan composition of a representative of the genus Rhodococcus as presently defined. These findings provide further chemotaxonomic evidence that lipoarabinomannan-type lipoglycans are widely distributed throughout the mycolic acid-containing actinomycetes.

Monoclonal antibodies reacting with the core oligosaccharide or lipid A component of Rhizobium lipopolysaccharide (LPS) could be useful for the elucidation of the structure and biosynthesis of this group of macromolecules. Mutant derivatives of Rhizobium leguminosarum 3841 with LPS structures lacking the major O-antigen moiety were used as immunogens, and eight antibodies were selected for further study. All the antibodies reacted with the fast-migrating species known as LPS-2 following gel electrophoresis of Rhizobium cell extracts. For four of these antibodies, reactivity with affinity-purified LPS was lost after mild acid hydrolysis, indicating that they probably recognized the core oligosaccharide component. The four other antibodies still reacted with acid-treated LPS and may recognize the lipid A moiety, which is stable to mild acid hydrolysis. The pattern of antibody staining after gel electrophoresis revealed differences in LPS-2 epitope structure between each of the mutants and the wild type. Furthermore, for each of the mutants the antibodies crossreacted with a minor band that migrated more slowly than LPS-2; we have termed this more slowly migrating form LPS-3. The majority of the antibodies also reacted with LPS from strain CE109, a derivative of Rhizobium etli CE3, confirming that the LPS core antigens can be relatively conserved between strains of different Rhizobium species. One of the antibodies isolated in this study (JIM 32) was unusual because it appeared to react with all forms of LPS from strain 3841 (namely, LPS-1, LPS-2, and LPS-3). Furthermore, JIM 32 reacted positively with the LPS from many strains of Rhizobium tested (excluding the Rhizobium meliloti subgroup). JIM 32 did not react with representative strains from Bradyrhizobium, Azorhizobium or other related bacterial species.

For identification of lipopolysaccharide (LPS)-associated epitopes of Legionella pneumophila serogroup 1, LPS of strain Philadelphia 1 was investigated using monoclonal antibodies (MAbs). The O-specific chain of LPS is a homopolymer of 5-acetamidino-7-acetamido-8-O-acetyl-3,5,7,9-tetradeoxy-D-glycero- L-galacto- nonulosonic acid. At least four immunoaccessible epitopes were recognized by different MAbs on the intact LPS. After O-deacetylation of LPS, the reactivity of one of the MAbs (MAb 3/1) was lost, indicating thus that the corresponding epitope is associated with the 8-O-acetyl group. Since the reactivity pattern of the MAb 3/1 is identical with those of the MAb 2 which was considered as a virulence marker for serogroup 1, this epitope may be involved in mediating virulence in L. pneumophila. Four MAbs specific to strains of serogroup 1 other than the monoclonal subtype Philadelphia recognized epitopes on the O-deacetylated LPS of strain Philadelphia 1 and, therefore, the virulence-associated epitope blocks recognition of the immunodeterminants that are accessible on the intact LPS of the strains lacking this epitope.

We have investigated the function of the Isi-1 gene of Neisseria gonorrhoeae previously implicated in lipopolysaccharide (LPS)-inner-core biosynthesis (Petricoin et al., 1991). Disruption of the gene in gonococcal strain MS11 resulted in the production of LPS that migrated faster than that from an isogenic galE mutant, typical for a mutation that influences the inner-core region. Complementation of a panel of Salmonella typhimurium mutants with defined defects in rfa loci demonstrated conclusively that the Isi-1 gene of MS11 is functionally homologous to the rfaF gene, which encodes heptosyltransferase II in both E. coli and S. typhimurium. Comparison of deduced amino acid sequences of the gonococcal and the Salmonella RfaF demonstrated 70% similarity, including 47% identical amino acid residues. Immunochemical analysis of the LPS using monoclonal antibodies directed against chemically defined inner-core glycoconjugates revealed that the gonococcal and Salmonella Rd2-chemotypes were antigenically similar, further extending the genetic and functional homology. Infection experiments in vitro demonstrated that the Isi-1 mutant could not invade human Chang epithelial cells despite expression of a genetically defined invasion-promoting gonococcal opacity protein. These data imply that the LPS phenotype is a critical factor for gonococcal invasiveness.

We have investigated the distribution of the various core types (R1, R2, R3, R4 and K-12) in 138 Escherichia coli isolates obtained from positive blood cultures. Rabbit antisera, raised against five rough strains expressing the respective core types, were made monospecific by extensive absorption. The reactivity of the antisera was tested in ELISA with bacterial cells that had been autoclaved for full exposure of core epitopes. One hundred and thirty strains could be typed directly, while eight strains required prior digestion with proteinase K for removal of cross-reactions. Ninety-four of the strains (68%) expressed the R1 type, and 9 (6.5%), 12 (8.7%), 7 (5.1%) and 3 (2.2%) strains expressed the R2, R3, R4 and K-12 core types, respectively. An R1R4 mixed core type, hitherto not yet described, was found in 13 (9.4%) strains. Results obtained with polyclonal antisera were in agreement with those obtained with monoclonal antibodies to the R1, R2 and R3 core types. Core typing may serve as an additional serological marker next to conventional typing of O-, H- and K-antigens.

Smooth, rough, and neutral forms of lipopolysaccharide (LPS) from Pseudomonas aeruginosa were used to assess the appropriate conditions for effective enzyme-linked immunosorbent assay (ELISA) of LPS. Each of these forms of well-defined LPS was tested for the efficiency of antigen coating by various methods as well as to identify an appropriate type of microtiter plate to use. For smooth LPS, the standard carbonate-bicarbonate buffer method was as efficient as the other sensitivity-enhancing plate-coating methods compared. The rough LPS, which has an overall hydrophobic characteristic, was shown to adhere effectively, regardless of the coating method used, to only one type of microtiter plate, CovaLink. This type of plate has secondary amine groups attached on its polystyrene surface by carbon chain spacers, which likely favors hydrophobic interactions between the rough LPS and the well surfaces. Dehydration methods were effective for coating microtiter plates with the neutral LPS examined, which is composed predominantly of a D-rhamnan. For the two dehydration procedures, LPS suspended in water or the organic solvent chloroform-ethanol was added directly to the wells, and the solvent was allowed to dehydrate or evaporate overnight. Precoating of plates with either polymyxin or poly-L-lysine did not give any major improvement in coating with the various forms of LPS. The possibility of using proteinase K- and sodium dodecyl sulfate-treated LPS preparations for ELISAs was also investigated. Smooth LPS prepared by this method was as effective in ELISA as LPS prepared by the hot water-phenol method, while the rough and neutral LPSs prepared this way were not satisfactory for ELISA.

We investigated the potency of various serotypes of lipopolysaccharides (LPS) by examining LPS-induced stimulation of PGI2 production and suppression of ACE activity in cultured human umbilical vein endothelial cells (HUVEC). HUVEC which had been incubated with E. coli 055:B5 and 0111:B4 for 24 h produced more prostacyclin (PGI2) in response to thrombin than HUVEC incubated with E. coli 026:B6. Also, angiotensin converting enzyme activity (ACE) in cell lysates of HUVEC incubated for 24 h with 055:B5 or 0111:B4 was suppressed significantly compared to control HUVEC or HUVEC incubated with 026:B6. From these experimental results, E. coli 055:B5 and 0111:B4 appear to be more potent than 026:B6. It is concluded that this difference in potency among various serotypes of LPS should be taken into account when experiments are designed to examine the effect of LPS on endothelial cell function.

We describe a method for the typing and quantitation of Klebsiella pneumoniae serotype O1 lipopolysaccharide (LPS) based on inhibition in an enzyme-linked immunosorbent assay of a reaction of known O1 LPS antigen and anti-O1 antibody by unknown LPS extracts. Serotype O1 was found in 32% of the 124 K. pneumoniae clinical isolates tested, showing that this serotype is frequent among the eight O serotypes which have been described previously.

Lipopolysaccharide from serostrains of Serpulina (Treponema) hyodysenteriae for serogroups A to I was characterized using sodium dodecylsulphate polyacrylamide gel electrophoresis and silver staining. All strains had lipopolysaccharide components ranging from 10 to 16 kDa that represented lipid A-core polysaccharide regions, and short O-antigen side chain were also recognized in certain immunoblots. Serological reactions between lipopolysaccharide and antisera against each of these serostrains were examined by Western immunoblotting. There was relatively little antigenic cross-reactivity between LPS from the nine strains, thus confirming their suitability as serostrains. Using cross-absorbed sera, isolates within serogroups A and E were shown to possess unique epitopes on the core lipopolysaccharide, distinct from serogroup reactivities. These isolates were therefore identified as serovars within the serogroups. This study confirmed the usefulness of the serotyping scheme for S. hyodysenteriae, in which the bacteria can be placed into serogroups using unabsorbed sera, and into serovars within these using cross-absorbed sera.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.

Out of 210 Proteus mirabilis isolates from bacteriuric patients a total of eight spontaneously agglutinating strains were found. SDS-PAGE analysis of their lipopolysaccharides (LPS), the separation of polysaccharide fractions (PS) by gel filtration, and chemical characterization of PSs were performed. Out of the eight strains one S form and one mutant classified as Rc were detected. The remaining six strains were recognized as Ra and intermediate forms. When tested in a hematogenous infection model in mice, the P. mirabilis Rc mutant survived in kidneys for at least two weeks, while the Re mutant used as control was eliminated within 20 h after the challenge. These data indicated that strains of P. mirabilis may be pathogenic even if they express very incomplete LPS.