Development of reagents and assays for the detection of pathogenic Burkholderia species.

Rapid detection of the category B biothreat agents Burkholderia pseudomallei and Burkholderia mallei in acute infections is critical to ensure that appropriate treatment is administered quickly to reduce an otherwise high probability of mortality (ca. 40% for B. pseudomallei). We are developing assays that can be used in clinical laboratories or security applications for the direct detection of surface-localized and secreted macromolecules produced by these organisms. We present our current medium-throughout approach for target selection and production of Burkholderia macromolecules and describe the generation of a Fab molecule targeted to the B. mallei BimA protein. We also present development of prototype assays for detecting Burkholderia species using anti-lipopolysaccharide antibodies.

Lipopolysaccharide from Burkholderia thailandensis E264 provides protection in a murine model of melioidosis.

Burkholderia thailandensis is a less virulent close relative of Burkholderia pseudomallei, a CDC category B biothreat agent. We have previously shown that lipopolysaccharide (LPS) extracted from B. pseudomallei can provide protection against a lethal challenge of B. pseudomallei in a mouse model of melioidosis. Sugar analysis on LPS from B. thailandensis strain E264 confirmed that this polysaccharide has a similar structure to LPS from B. pseudomallei. Mice were immunised with LPS from B. thailandensis or B. pseudomallei and challenged with a lethal dose of B. pseudomallei strain K96243. Similar protection levels were observed when either LPS was used as the immunogen. This data suggests that B. thailandensis LPS has the potential to be used as part of a subunit based vaccine against pathogenic B. pseudomallei.

Protective response to subunit vaccination against intranasal Burkholderia mallei and B. pseudomallei challenge.

Burkholderia mallei and B. pseudomallei are Gram-negative pathogenic bacteria, responsible for the diseases glanders and melioidosis, respectively. Furthermore, there is currently no vaccine available against these Burkholderia species. In this study, we aimed to identify protective proteins against these pathogens. Immunization with recombinant B. mallei Hcp1 (type VI secreted/structural protein), BimA (autotransporter protein), BopA (type III secreted protein), and B. pseudomallei LolC (ABC transporter protein) generated significant protection against lethal inhaled B. mallei ATCC23344 and B. pseudomallei 1026b challenge. Immunization with BopA elicited the greatest protective activity, resulting in 100% and 60% survival against B. mallei and B. pseudomallei challenge, respectively. Moreover, sera from recovered mice demonstrated reactivity with the recombinant proteins. Dendritic cells stimulated with each of the different recombinant proteins showed distinct cytokine patterns. In addition, T cells from immunized mice produced IFN-γ following in vitro re-stimulation. These results indicated therefore that it was possible to elicit cross-protective immunity against both B. mallei and B. pseudomallei by vaccinating animals with one or more novel recombinant proteins identified in B. mallei.

Phosphoglycerate kinase 2 (PGK2) is essential for sperm function and male fertility in mice.

Phosphoglycerate kinase 2 (PGK2), an isozyme that catalyzes the first ATP-generating step in the glycolytic pathway, is encoded by an autosomal retrogene that is expressed only during spermatogenesis. It replaces the ubiquitously expressed phosphoglycerate kinase 1 (PGK1) isozyme following repression of Pgk1 transcription by meiotic sex chromosome inactivation during meiotic prophase and by postmeiotic sex chromatin during spermiogenesis. The targeted disruption of Pgk2 by homologous recombination eliminates PGK activity in sperm and severely impairs male fertility, but does not block spermatogenesis. Mating behavior, reproductive organ weights (testis, excurrent ducts, and seminal vesicles), testis histology, sperm counts, and sperm ultrastructure were indistinguishable between Pgk2(-/-) and wild-type mice. However, sperm motility and ATP levels were markedly reduced in males lacking PGK2. These defects in sperm function were slightly less severe than observed in males lacking glyceraldehyde-3-phosphate dehydrogenase, spermatogenic (GAPDHS), the isozyme that catalyzes the step preceding PGK2 in the sperm glycolytic pathway. Unlike Gapdhs(-/-) males, the Pgk2(-/-) males also sired occasional pups. Alternative pathways that bypass the PGK step of glycolysis exist. We determined that one of these bypass enzymes, acylphosphatase, is active in mouse sperm, perhaps contributing to phenotypic differences between mice lacking GAPDHS or PGK2. This study determined that PGK2 is not required for the completion of spermatogenesis, but is essential for sperm motility and male fertility. In addition to confirming the importance of the glycolytic pathway for sperm function, distinctive phenotypic characteristics of Pgk2(-/-) mice may provide further insights into the regulation of sperm metabolism.

Formation of stable submicron protein particles by thin film freezing.

PURPOSE: Highly stable, submicron lactate dehydrogenase (LDH) and lysozyme particles may be produced by thin film freezing (TFF) of aqueous solutions followed by lyophilization. METHODS: The LDH activity was determined by measuring the decrease in absorbance of NADH over time for the reaction of pyruvate to lactate. For lysozyme the particle morphology was determined by scanning electron microscopy (SEM) and compared with the specific surface area (BET) and the particle size, as measured by laser light scattering, RESULTS: Protein particles with an average diameter of 300 nm and 100% enzyme activity upon reconstitution (for LDH) were formed by TFF. Droplets of protein solutions, 3.6 mm in diameter, spread upon impact with 223 and 133 K metal surfaces to form cylindrical disks with thicknesses of 200-300 microm. Calculated cooling rates of the disks of 10(2) K/s were confirmed experimentally with infrared measurements. CONCLUSIONS: The cooling rates of 10(2) K/s, intermediate to those in lyophilization (1 K/min) and spray freeze-drying (SFD) (10(6) K/s), were sufficiently fast to produce sub-micron protein particles with surface areas of 31-73 m2/g, an order of magnitude higher than in lyophilization. In addition, the low surface area/volume ratio (32-45 cm(-1)) of the gas-liquid interface led to minimal protein adsorption and denaturation relative to SFD.

Development of aptamers specific for potential diagnostic targets in Burkholderia pseudomallei.

Improved diagnostic reagents would be of considerable benefit in enhancing the specificity and sensitivity of rapid assays for Burkholderia pseudomallei, the causative agent of melioidosis. The purpose of this work is to develop aptamers, high affinity RNA-based molecular recognition molecules, which could be used as reagents for identification of the whole organism in assays of biological samples. Data are presented demonstrating the purification of recombinant B. pseudomallei secreted or surface-exposed macromolecules, which have been expressed in Escherichia coli, and the initial stages of aptamer generation using these recombinant proteins. Future studies will focus upon the expansion of this methodology to include other target macromolecules located on or near the outer membrane of this organism.

Sero-characterization of lipopolysaccharide from Burkholderia thailandensis.

We report the successful purification of lipopolysaccharide (LPS) from Burkholderia thailandensis, a Gram-negative bacterium, closely related to the highly pathogenic organisms B. pseudomallei and B. mallei. Burkholderia thailandensis LPS is shown to cross-react with rabbit and mouse sera obtained from inoculation with B. pseudomallei or B. mallei, respectively. These data suggest that B. thailandensis LPS shares similar structural features with LPS molecules from highly pathogenic Burkholderia species. This information may prove useful in ongoing efforts to develop novel vaccines and/or diagnostic reagents.

In silico analysis of potential diagnostic targets from Burkholderia pseudomallei.

Administration of appropriate therapeutic regimes for infections arising from pathogenic species of Burkholderia is critically dependent upon rapid and accurate diagnoses. The purpose of this work is to establish a bioinformatic pipeline to assess protein sequences for their potential as diagnostic targets for the detection of Burkholderia species. Data are presented showing both a bioinformatic methodology for prediction of surface-associated and secreted proteins and its application to a test dataset of proteins from the pathogen B. pseudomallei. A subset of proteins, known to be produced by the organism, is identified which represents potential targets for development of new diagnostic reagents. In addition, a 'reverse diagnostics' bioinformatics approach has been established which can now be extended to whole genome analyses.