Burkholderia pseudomallei and B. mallei are the etiological agents of the diseases melioidosis and Glanders, respectively. They are related Gram-negative bacterial pathogens affecting both human and animal hosts. B. pseudomallei and B. mallei are infectious through both subcutaneous inoculation and inhalation routes of entry and both pathogens can elicit septic infections with very high mortality rates (>80%). Since both pathogens could be used as biological agents against naive human populations, they have been designated as Category B Biological Agents by the Centers for Disease Control (CDC). Widespread dissemination of infectious strains would require an aerosolized delivery for maximum impact, and therefore, understanding the pulmonary forms of glanders and melioidosis is much more important than studying the subcutaneous forms of these diseases. As such, defining the bacteria/host interactions that lead to pulmonary pathology has become a major focus of our laboratory. B. pseudomallei and B. mallei are facultative intracellular pathogens that can invade, survive and replicate in epithelial and phagocytic cell lines. In order to avoid clearance by phagocytic cells, B. pseudomallei and B. mallei possess an extensive repertoire of oxidative/nitrositive stress proteins (e.g., SOD, catalase, glutathione peroxidase etc.) that reduce/eliminate toxic oxygen/nitrogen radicals generated by the host. In E. coli, expression of similar defense enzymes is regulated by the transcriptional activator, OxyR. Mutants in B. mallei ATCC23344 and B. pseudomallei DD503 have been constructed by insertional inactivation via conjugative transfer of the plasmid pZSV. The resulting delta-oxyR mutants were more sensitive to hydrogen peroxide compared to the wild-type, parent strains. Using a modified kanamycin-protection assay, B. pseudomallei oxyR mutants were unable to survive in mouse or human macrophages and had decreased survivability in mouse or human epithelial cells. In addition, the B. mallei oxyR mutant was unable to survive in primary and immortal mouse macrophage cells. To further assess the role of OxyR in pathogenesis, mice were challenged with wild-type (WT) B. pseudomallei, WT B. mallei or oxyR mutants. As suggested by the macrophage assays, oxyR mutants of both B. mallei and B. pseudomallei were non-infectious, suggesting a critical role for OxyR in virulence of these pathogens. These experiments are providing valuable insight into the role of the oxidative stress response in the survival and pathogenesis of Burkholderia sp. Several animal models have been used for the study of B. pseudomallei and B. mallei. The current animal model used for studying both melioidosis and glanders by laboratories worldwide is the murine model. Since the mouse can be used as a model for both chronic and acute forms of these diseases, is cost effective and has excellent genetic tools available, it is ideal for assesing critical aspects of burkholderia pathogenesis and the host response to colonization, infection and disease. A focus of our laboratory has been the development of acute murine respiratory melioidosis and glanders models of infection. Mice infected by the typical intranasal route of infection develop multifocal lesions of the lung as well as inflammation of the tracheobronchial lymph node, upper respiratory tract, and inflammation blood-brain barrier (meningitis). In currently used mouse models, neurological symptoms associated with meningitis are a common complication necessitating the euthanization of animals before acute respiratory melioidosis occurs. To overcome this problem, our lab has refined a technique for intratracheal inoculation which allows for direct insertion of the bacteria into the lungs of the mouse while maintaining BSL-3 containment. Further, we have introduced the Photorhabdus lux operon into pathogenic B. pseudomallei in order of visulalize bacteria during the infection of live animals. Diagnostic imaging, used to monitor bacterial colonization of mouse target organs (i.e., lungs, liver, and spleen), showed that mice inoculated using the improved intratracheal technique developed pulmonary lesions after 48h. However, these infected animals did not develop the upper respiratory and meningial infections typical observed when intranasal infection methods are used. Additionally, this technique has allowed us to characterize mutants and identify important genes involved in respiratory melioidosis. Using this technique, we have demonstrated that B. mallei and B. pseudomallei strains lacking a functional OxyR are avirulent. In addition, strains lacking the alternative sigma factor, RpoN or the nitrositive stress response regulator, NsrR, have an attenuated virulence phenotype. Based on these data, it appears that the intratracheal infection technique that we have developed allows for the direct delivery of the pathogen to the lungs and more closely mimics pulmonary melioidosis. This will be a very helpful tool for idenifying genes encoding proteins involved in the pathogenesis of acute pulmonary melioidosis and Glanders.
