Biological activities of lipopolysaccharide of Burkholderia (Pseudomonas) pseudomallei

Abstract Endotoxic activities of lipopolysaccharide (LPS) isolated from Burkholderia (Pseudomonas) pseudomallei , a causative agent of melioidosis, were investigated. Compared to an enterobacterial LPS (SAE-LPS), B. pseudomallei LPS (BP-LPS) exhibited weaker pyrogenic activity in rabbits, lethal toxicity in galactosamine-sensitized mice and murine macrophage activation, i.e. production of tumor necrosis factor, interleukin-6 and nitric oxide. BP-LPS, on the other hand, exhibited stronger mitogenic activity to murine splenocytes than SAE-LPS; moreover, it stimulated even the splenocytes of LPS-resistant C3H/HeJ mice. Unusual chemical structures in the acid-stable inner core region attached to the lipid A moiety of BP-LPS may be responsible for this strong mitogenic activity.     

Laboratory Investigation of Ecological Factors Influencing the Environmental Presence of Burkholderia pseudomallei

Factors like temperature, pH value, water content in soil, and ultraviolet rays that might have influence on the survival of environmental Burkholderia pseudomallei strains were evaluated. Data showed that the optimal temperature and pH value for B. pseudomallei were 24 C to 32 C and 5 to 8, respectively. Water content in soil of less than 10% brought about the death of the bacteria within 70 days, while water content of more than 40% maintained bacteria life for 726 days. The bacteria were easily killed by ultraviolet rays at 465 μW/cm² for 7.75 min while other permanent soil bacteria were killed at 1,860 μW/cm² for 31 min. From these results, it could be concluded that proper temperature, enough water in soil, and suitable soil pH might be the three major ecological conditions governing the environmental presence of B. pseudomallei.     

Extraction and characterization of lipopolysaccharide from Pseudomonas pseudomallei

The best yield of lipopolysaccharide (LPS) of Pseudomonas pseudomallei GIFU 12046 was obtained by extraction of defatted cells by phenol/chloroform/petroleum ether. The LPS showed a smooth character on SDS-polyacrylamide gel electrophoresis and contained D-glucose, L-glycero-D-manno-heptose, and D-glucosamine as the main sugar components, and 3-hydroxypalmitic acid as an amide-linked fatty acid. The growth conditions did not affect the electrophoresis profile and chemical composition of LPS. 2-Keto-3-deoxyoctonic acid was not detectable, and mild acid hydrolysis could not liberate free lipid A, suggesting that the linkage between inner core and lipid A was stable against acid hydrolysis, and the structure of this region is similar to that of P. cepacia, which has close taxonomic relationship with P. pseudomallei.     

Characterization of three capsular polysacharides produced by Burkholderia pseudomallei

Three kinds of capsular polysaccharide (CP) were found to be produced by Burkholderia pseudomallei. When the bacterium was grown with the medium without glycerol, CP-1a and CP-1b were produced. CP-1a was mainly 1.4-linked glucan and CP-1b was identified as a polymer composed of galactose and 3-deoxy-D-manno-octulosonic acid, whose chemical structure was recently reported by other laboratories. When the bacterium was grown with the medium containing 5" glycerol. CP-2 was synthesized. CP-2 contained galactose, rhamnose, mannose, glucose and a uronic acid in a ratio of approximately 3:1:0.3:1:1. Methylation analysis of the purified polysaccharides demonstrated that the two acidic polysaccharides. CP-1b and CP-2 shared no common structure, indicating that CP-2 was an acidic capsular polysaccharide whose chemical characters were not reported previously.     

Phenotypic characteristics and pathogenic ability across distinct morphotypes of Burkholderia pseudomallei DT

Burkholderia pseudomallei DT is unusual as it exhibits six distinct colony morphotypes. Types III and V show stronger motility, whereas type VI exhibits the highest levels of bacterial association with peritoneal exudate cells. Although the bacterial loads in the organs are not significantly different for infections by the six distinct morphotypes, higher mortality (100% and 89%, respectively) and larger areas of abnormal liver debris (20.6% and 22.4%, respectively) are found with types I- and III-infected mice compared to the others. These morphotypes sometimes undergo switching to a mucoid type in the body of mice, but the reverse has never been observed.     

Genome-wide expression analysis of iron regulation in Burkholderia pseudomallei and Burkholderia mallei using DNA microarrays

Burkholderia pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. As iron regulation of gene expression is common in bacteria, in the present studies, we have used microarray analysis to examine the effects of growth in different iron concentrations on the regulation of gene expression in B. pseudomallei and B. mallei. Gene expression profiles for these two bacterial species were similar under high and low iron growth conditions irrespective of growth phase. Growth in low iron led to reduced expression of genes encoding most respiratory metabolic systems and proteins of putative function, such as NADH-dehydrogenases, cytochrome oxidases, and ATP-synthases. In contrast, genes encoding siderophore-mediated iron transport, heme-hemin receptors, and a variety of metabolic enzymes for alternative metabolism were induced under low iron conditions. The overall gene expression profiles suggest that B. pseudomallei and B. mallei are able to adapt to the iron-restricted conditions in the host environment by up-regulating an iron-acquisition system and by using alternative metabolic pathways for energy production. The observations relative to the induction of specific metabolic enzymes during bacterial growth under low iron conditions warrants further experimentation.     

Burkholderia multivorans acts as an antagonist against the growth of Burkholderia pseudomallei in soil

In this study, it was demonstrated, by using agar diffusion tests and a Transwell system, that Burkholderia multivorans NKI379 has an antagonistic effect against the growth of B. pseudomallei. Bacterial representatives were isolated from agricultural crop soil and mixed to construct a partial bacterial community structure that was based on the results of reproducible patterns following PCR-denaturing gradient gel electrophoresis analysis of total soil chromosomes. The antagonistic effect of B. multivorans on B. pseudomallei was observed in this imitate community. In a field study of agricultural crop soil, the presence of B. pseudomallei was inversely related to the presence of the antagonistic strains B. multivorans or B. cenocepacia. B. multivorans NKI379 can survive in a broader range of pH, temperatures and salt concentrations than B. pseudomallei, suggesting that B. multivorans can adapt to extreme environmental changes and therefore predominates over B. pseudomallei in natural environments.     

Antigenic relatedness between Burkholderia pseudomallei and Burkholderia mallei

Burkholderia pseudomallei and Burkholderia mallei are causative agents of distinct diseases, namely, melioidosis and glanders, respectively. The two species are very closely related, based on DNA-DNA homology, base sequence of the 16S rRNA, and phenotypic characteristics. Based on the use of polyclonal antisera, B. pseudomallei and B. mallei are also found to be antigenically closely related to one another. We previously reported the production of monoclonal antibodies (MAbs) against B. pseudomallei antigens; one group was specific for the 200-kDa exopolysaccharide present on the surface of all B. pseudomallei isolates, and the other was specific for the lipopolysaccharide (LPS) structure present on more than 95% of the B. pseudomallei tested. In the present study, we showed that the MAbs against 200-kDa antigen of B. pseudomallei cross-reacted with a component present also in some B. mallei isolates (3/6), but the positive immunoblot reaction was noted below the 200-kDa position. On the other hand, none of the six B. mallei isolates reacted with the MAb specific for B. pseudomallei LPS. It was of interest to observe that only the 3 exopolysaccharide-positive B. mallei isolates reacted with a commercial MAb against B. mallei LPS. The data presented suggest that B. mallei can be classified antigenically into two types based on their reactivities with different MAbs, i.e., the presence or absence of exopolysaccharide and the types of lipopolysaccharide. The heterogeneity of the LPS from these two closely related organisms is most likely related to the differences in its O-polysaccharide side chain.     

The molecular and cellular basis of pathogenesis in melioidosis: how does Burkholderia pseudomallei cause disease?

Melioidosis, a febrile illness with disease states ranging from acute pneumonia or septicaemia to chronic abscesses, was first documented by Whitmore & Krishnaswami (1912) . The causative agent, Burkholderia pseudomallei , was subsequently identified as a motile, gram-negative bacillus, which is principally an environmental saprophyte. Melioidosis has become an increasingly important disease in endemic areas such as northern Thailand and Australia ( Currie et al. , 2000 ). This health burden, plus the classification of B. pseudomallei as a category B biological agent ( Rotz et al. , 2002 ), has resulted in an escalation of research interest. This review focuses on the molecular and cellular basis of pathogenesis in melioidosis, with a comprehensive overview of the current knowledge on how B. pseudomallei can cause disease. The process of B. pseudomallei movement from the environmental reservoir to attachment and invasion of epithelial and macrophage cells and the subsequent intracellular survival and spread is outlined. Furthermore, the diverse assortment of virulence factors that allow B. pseudomallei to become an effective opportunistic pathogen, as well as to avoid or subvert the host immune response, is discussed. With the recent increase in genomic and molecular studies, the current understanding of the infection process of melioidosis has increased substantially, yet, much still remains to be elucidated.     

类鼻疽伯克霍尔德菌感染与免疫逃逸机制的研究进展

类鼻疽是由类鼻疽伯克霍尔德菌(Burkholderia pseudomallei,B. pseudomallei)(简称类鼻疽菌)感染引起的一种热带医学疾病。该病临床表现复杂多样,严重感染时可快速发展为败血症,病死率高达40%。越来越多的证据表明,它是一种正在扩散的人兽共患传染病。本文就近年来关于类鼻疽菌感染的重要毒力因子以及其在免疫逃逸中的作用机制研究进展进行总结,以期了解类鼻疽菌的致病机制,为将来有效疫苗和治疗药物的研发提供理论指导。     

Antigenic Differences between Clinical and Environmental Isolates of Burkholderia pseudomallei

Burkholderia pseudomallei is a free-living organism that causes the potentially lethal tropical infection melioidosis. The disease is endemic in many parts of eastern Asia and northern Australia. The presence of two distinct biotypes in soil can be reliably distinguished by their ability to assimilate l -arabinose. Whereas some soil isolates could utilize this substrate (Ara⁺), the remaining soil isolates and all clinical isolates tested so far could not (Ara^?). Only the Ara^? isolates were virulent in animal models. We have raised a murine monoclonal antibody (MAb) that can readily distinguish Ara^? from Ara⁺ biotypes. The MAb reacted with a high molecular weight component present only on the Ara^? biotype. With this MAb, clinical and soil Ara^?isolates gave identical positive reactions in agglutination, immunofluorescence, ELISA and immunoblot assays. Using these same assay systems, the soil Ara⁺ biotype did not react with the MAb. Similar but distinct immunoblot patterns were also noted when these two Ara biotypes were probed with sera from patients with melioidosis or with polyclonal immune rabbit sera. These data showed that the Ara^? biotype from both clinical and environmental isolates is antigenically different from its Ara⁺ environmental counterpart. The SDS-PAGE protein and lectin-binding profiles of both groups of Ara^? isolates were also found to be different from those of the Ara⁺ biotype.     

Neutralization of Burkholderia pseudomallei Protease by Fabs Generated through Phage Display

The isolation of therapeutic and functional protease inhibitors in vitro via combinatorial chemistry and phage display technology has been described previously. Here we report the construction of a combinatorial mouse-human chimeric antibody fragment (Fab) antibody library targeted against the protease of the tropical pathogen, Burkholderia pseudomallei. The resulting library was biopanned against the protease, and selected clones were analyzed for their ability to function as protease inhibitors. Three families of Fabs were identified by restriction fingerprinting, all of which demonstrated high specificity towards the protease of B. pseudomallei. Purified Fabs also demonstrated the capacity to inhibit B. pseudomallei protease activity in vitro, and this inhibitory property was exclusive to the pathogenic protease. Thus these recombinant antibodies are candidates for immunotherapy and tools to aid in further elucidation of the mechanism of action of the B. pseudomallei protease.     

Separation of 6-deoxy-heptan [correction of 6-deoxy-heptane] from a smooth-type lipopolysaccharide preparation of Burkholderia pseudomallei

Smooth-type lipopolysaccharide (LPS) of Burkholderia pseudomallei has been reported to contain two kinds of O-antigenic polysaccharides, a 1,3-linked homopolymer of 6-deoxy-heptose and a polymer with a repeating unit of -->3)-glucose-(1-->3)-6-deoxy-talose-(1--> with O-acetyl or O-methyl modifications. A LPS preparation containing these two polysaccharides was separated by gel-permeation chromatography in this study. Chemical analysis of the separated fractions revealed the 6-deoxy-heptan [corrected] to be a polysaccharide without a lipid portion and the polymer of glucose and 6-deoxy-talose to be an O-antigenic polysaccharide of the LPS. This result was further supported by the assay of these polysaccharide molecules for macrophage activation activity. The 6-deoxy-heptan [corrected] showed no macrophage activation, indicating that this polysaccharide was not the LPS, but one of the capsular polysaccharides of B. pseudomallei.     

Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice

Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1). They were still 50-80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1). The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1). Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.     

Epitope mapping of Burkholderia pseudomallei serine metalloprotease: identification of serine protease epitope mimics

Filamentous phage random peptide libraries were used to identify the epitopes of Burkholderia pseudomallei protease by panning against IgG polyclonal sera that exhibited protease neutralizing properties. The isolated fusion peptides presented a consensus peptide sequence, TKSMALSG, which closely resembles part of the active site sequence, 435GTSMATPHVAG445, of B. pseudomallei serine metalloprotease. By comparing the consensus sequence, TKSMALSG, with the predicted three-dimensional molecular model of B. pseudomallei serine metalloprotease, it appears that the potential antibody binding epitope was buried within the molecule. This active site was conformational whereby one continuous sub-region (SMA) was located between two discontinuous sub-regions, supplied by the flanking residues in the same polypeptide. All phages selected from the biopanning with IgG polyclonal sera showed good binding towards the polyclonal antibodies when compared to the negative control. In addition, these peptide-bearing phages showed competitive inhibition of B. pseudomallei serine metalloprotease binding to the polyclonal IgG.     

Detection and differentiation of Burkholderia pseudomallei, Burkholderia mallei and Burkholderia thailandensis by multiplex PCR

Burkholderia pseudomallei, a Gram-negative bacterium that causes melioidosis may be differentiated from closely related species of Burkholderia mallei that causes glanders and non-pathogenic species of Burkholderia thailandensis by multiplex PCR. The multiplex PCR consists of primers that flank a 10-bp repetitive element in B. pseudomallei and B. mallei amplifying PCR fragment of varying sizes between 400-700 bp, a unique sequence in B. thailandensis amplifying a PCR fragment of 308 bp and the metalloprotease gene amplifying a PCR fragment of 245 bp in B. pseudomallei and B. thailandensis. The multiplex PCR not only can differentiate the three Burkholderia species but can also be used for epidemiological typing of B. pseudomallei and B. mallei strains.     

Comparison of the amine/amino acid activation profiles of the β- and γ-carbonic anhydrases from the pathogenic bacterium Burkholderia pseudomallei

The β-class carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium Burkholderia pseudomallei, BpsCAβ, that is responsible for the tropical disease melioidosis was investigated for its activation with natural and non-natural amino acids and amines. Previously, the γ-CA from this bacterium has been investigated with the same library of 19 amines/amino acids, which show very potent activating effects on both enzymes. The most effective BpsCAβ activators were L- and D-DOPA, L- and D-Trp, L-Tyr, 4-amino-L-Phe, histamine, dopamine, serotonin, 2-pyridyl-methylamine, 1-(2-aminoethyl)-piperazine and L-adrenaline with K_As of 0.9–27?nM. Less effective activators were D-His, L- and D-Phe, D-Tyr, 2-(2-aminoethyl)pyridine and 4-(2-aminoethyl)-morpholine with K_As of 73?nM–3.42?µM. The activation of CAs from bacteria, such as BpsCAγ/β, has not been considered previously for possible biomedical applications. It would be of interest to perform studies in which bacteria are cultivated in the presence of CA activators, which may contribute to understanding processes connected with the virulence and colonization of the host by pathogenic bacteria.