Overview
Burkholderia pseudomallei is a soil-dwelling Gram-negative bacterium and the causative agent of melioidosis. Endemic to southeast Asia, northern Australia, and other parts of the globe, B. pseudomallei infects its hosts through cuts, inhalation, and ingestion from environmental sources such as rice fields and contaminated water supplies. Melioidosis is notoriously difficult to treat, even if properly diagnosed. B. pseudomallei has earned the nickname “The Great Mimicker” for its ability to fool clinicians into categorizing cases of melioidosis as cases of tuberculosis, preventing timely treatment for the disease. B. pseudomallei is highly antibiotic-resistant and can carry a mortality rate of > 50% even with appropriate treatment. Inoculation with B. pseudomallei can rapidly progress from pneumonia or ulceration to bacteremia and severe sepsis. Chronic and latent infections also occur, with the highest reported incubation period being 62 years with its ability to persist in a host poorly characterized. B. pseudomallei is a pressing threat to global health and biosecurity that requires a swift response to characterize its pathogenesis and ecology and develop new therapeutic countermeasures.
Pathogenesis
As an intracellular pathogen, Burkholderia pseudomallei must be able to invade the host cell, avoid phagosomal destruction by the host, replicate intracellularly, and spread to nearby cells to start the cycle again. Unlike other intracellular pathogens such as Listeria monocytogenes, Burkholderia pseudomallei intercellular spread is facilitated by fusion of the host cell with nearby cells, resulting in the formation of Multi-Nuclear Giant Cells (MNGCs) which eventually lyse to form a plaque. The formation of MNGCs is a hallmark of B. pseudomallei pathogenesis and does not occur without the presence of a functional Type VI Secretion System. We are identifying the specific components of the Type VI Secretion System required for host cell fusion and the association between fusogenic activity and virulence.
Ecology
Ongoing efforts to define the environmental conditions of melioidosis-endemic regions have focused primarily on climatic and geochemical properties. Aside from a few broad correlations with rainfall and soil type, these investigations have not been very productive.Thus, a greater understanding of melioidosis etiology will only be gained by investigations into its ecology. We are evaluating the ecological species relationships and interactions of Burkholderia pseudomallei (Bp) as predictors of endemic potential. Our central hypothesis is that virulence traits in Bp evolved for defense against environmental predators, and as a mechanism for survival within host reservoir species. Accordingly, virulence loci are maintained by positive selection though interactions with co-endemic fauna, and facilitate survival in the environment by conserved mechanisms that also promote mammalian disease. We are identifying and characterizing environmental factors and the relationships between Bp and other ecosystem species, and exploring how these affect the presence and virulence of Bp, and consequently, the incidence of disease in humans and mammals. The aim of Task 1 is to investigate relationships between environmental conditions, strain diversity, and virulence in mammals. We are using multiplex, high-throughput whole genome sequencing (HT-WGS) for polymorphism analysis of large numbers of strains. Sequenced strains exhibiting polymorphic virulence loci are tested for their ability to cause disease in mice. In Task 2, we employ parallel HTS metagenomic and metatranscriptomic analysis to identify predictors of endemicity as “signatures” of co-endemic fauna, followed by investigations of their associations with virulent Bp genotypes. The objective of Task 3 is to define the nature of various interactions between Bp and fauna. We are isolating macrofauna (insects, large larvae and nematodes), mesofauna (mites, ants, fungal hyphae, small larvae and nematodes) and microfauna (bacteria and protozoa) from environmental specimens using a differential fractionation and sorting scheme. Using virulent Bp or non-virulent E. coli as “bait”, we are isolating predator and host organisms by their differential susceptibilities to Bp lethality mechanisms, and performing species identification by metabarcoded amplification and HTS. This allows us to evaluate ecological species for their utility in biocontrol or diagnostic surveillance prediction. Ultimately, we hope to compare ecological signatures from geographically separate regions, incorporate our findings into a global map of Bp endemicity, and apply our biocontrol strategies to eradicate Bp from endemic soil.
Therapeutics
Even with appropriate treatment, melioidosis has a mortality rate of 54% (Limmathurotsakul et al., 2016). We are developing a robust vaccine and new effective treatment options in order to counter the extraordinarily high mortality rate and high level of intrinsic antibiotic resistance in B. pseudomallei.
Prevention
There is currently no FDA-approved vaccine for the prevention of melioidosis, which presents in both acute and chronic forms. We are collaborating with Dr. Leonard Rome of the California NanoSystems Institute to develop a vaccine delivery system using vault nanoparticles.
Treatment
In collaboration with Dr. Jeff F. Miller of the California NanoSystems Institute, we have identified a novel small molecule inhibitor of Burkholderia pseudomallei intracellular spread, Burkfloxacin, and found that a pre-existing antifungal medication, 5-fluorocytosine (5-FC), also inhibits intercellular spread. Given that repurposing pre-existing medications does not require a lengthy approval process, our efforts are currently focused on 5-FC. We are seeking to 1) identify the specific mechanism by which 5-FC prevents intercellular spread 2) identify the effects of 5-FC resistance on B. pseudomallei pathogenesis and 3) explore the efficacy of combining 5-FC with other antibiotics to treat B. pseudomallei infections.