Enzyme-linked immunosorbent assay (ELISA) using a glycolipid antigen for the serodiagnosis of melioidosis

Abstract The serodiagnosis of melioidosis is commonly performed with tests using protein or polysaccharide as antigen. However, due to the low sensitivity, specificity and difficulty in the preparation of the antigens, more simple, precise and reproducible diagnostic tests were required. A purified glycolipid antigen (GL) which is a specific lipid component of Burkholderia pseudomallei has been used in an ELISA. With this antigen, specific immunoglobulin G (IgG) was detected in 49 out of 50 melioidosis sera. IgG was also detected in 2 out of 185 (Japanese) and 16 out of 181 (Vietnamese) control sera. Thus, the sensitivity was 98.0%, and specificity was 98.9% and 91.1% in the Japanese and Vietnamese sera, respectively. When the ELISA and indirect haemagglutination (IHA) tests were combined, a sensitivity of 100% and specificity of 97.8% were achieved. The advantages of the glycolipid antigen are ease of preparation, stability, high sensitivity and specificity.     

Identification of new transposable genetic elements in Burkholderia pseudomallei using subtractive hybridisation

A subtraction library of Burkholderia pseudomallei was constructed by subtractive hybridisation of B. pseudomallei genomic DNA with Burkholderia thailandensis genomic DNA. Two clones were found to have significant sequence similarity to insertion sequences which have previously not been found in B. pseudomallei (designated ISA and ISB); and two clones showed sequence similarity to different regions of Burkholderia cepacia IS407 that has recently been detected in B. pseudomallei. The former, though possibly non-functional, represents new transposable genetic elements of B. pseudomallei. All three sequences were found to be present in multi-copy in the genomes of a number of B. pseudomallei strains and in B. thailandensis, which are the first transposable elements identified in this species.     

AFN-1252 is a potent inhibitor of enoyl-ACP reductase from Burkholderia pseudomallei—Crystal structure,mode of action,and biological activity

Melioidosis is a tropical bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei; Bpm), a Gram-negative bacterium. Current therapeutic options are largely limited to trimethoprim-sulfamethoxazole and β-lactam drugs, and the treatment duration is about 4 months. Moreover, resistance has been reported to these drugs. Hence, there is a pressing need to develop new antibiotics for Melioidosis. Inhibition of enoyl-ACP reducatase (FabI), a key enzyme in the fatty acid biosynthesis pathway has shown significant promise for antibacterial drug development. FabI has been identified as the major enoyl-ACP reductase present in B. pseudomallei. In this study, we evaluated AFN-1252, a Staphylococcus aureus FabI inhibitor currently in clinical development, for its potential to bind to BpmFabI enzyme and inhibit B. pseudomallei bacterial growth. AFN-1252 stabilized BpmFabI and inhibited the enzyme activity with an IC₅₀ of 9.6 nM. It showed good antibacterial activity against B. pseudomallei R15 strain, isolated from a melioidosis patient (MIC of 2.35 mg/L). X-ray structure of BpmFabI with AFN-1252 was determined at a resolution of 2.3 Å. Complex of BpmFabI with AFN-1252 formed a symmetrical tetrameric structure with one molecule of AFN-1252 bound to each monomeric subunit. The kinetic and thermal melting studies supported the finding that AFN-1252 can bind to BpmFabI independent of cofactor. The structural and mechanistic insights from these studies might help the rational design and development of new FabI inhibitors.     

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.     

Deficiency of protease-activated receptor-1 limits bacterial dissemination during severe Gram-negative sepsis (melioidosis)

Protease-activated receptor-1 (PAR-1) is a G-coupled transmembrane receptor expressed by multiple cell types present in the lungs that can be activated by various proteases generated during acute inflammation. In this study we aimed to investigate the role of PAR-1 during melioidosis, a common cause of (pneumo)sepsis in Southeast Asia in a murine model of intranasal inoculation of the causative pathogen Burkholderia (B.) pseudomallei. Our results show that endogenous PAR-1 facilitates bacterial growth and dissemination during murine melioidosis, which is associated with increased cell influxes. However, these observations have no impact on survival.     

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.     

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.