Dictyostelium discoideum as a Model System for Identification of Burkholderia pseudomallei Virulence Factors

Abstract

Burkholderia pseudomallei is an emerging bacterial pathogen and category B biothreat. Human infections with B. pseudomallei (called melioidosis) present as a range of manifestations, including acute septicemia and pneumonia. Although melioidosis can be fatal, little is known about the molecular basis of B. pseudomallei pathogenicity, in part because of the lack of simple, genetically tractable eukaryotic models to facilitate en masse identification of virulence determinants or explore host-pathogen interactions. Two assays, one highthroughput and one quantitative, were developed to monitor levels of resistance of B. pseudomallei and the closely related nearly avirulent species Burkholderia thailandensis to predation by the phagocytic amoeba Dictyostelium discoideum. The quantitative assay showed that levels of resistance to, and survival within, amoeba by these bacteria and their known virulence mutants correlate well with their published levels of virulence in animals. Using the high-throughput assay, we screened a 1,500-member B. thailandensis transposon mutant library and identified 13 genes involved in resistance to predation by D. discoideum. Orthologs of these genes were disrupted in B. pseudomallei, and nearly all mutants had similarly decreased resistance to predation by D. discoideum. For some mutants, decreased resistance also correlated with reduced survival in and cytotoxicity toward macrophages, as well as attenuated virulence in mice. These observations suggest that some factors required by B. pseudomallei for resistance to environmental phagocytes also aid in resistance to phagocytic immune cells and contribute to disease in animals. Thus, D. discoideum provides a novel, highthroughput model system for facilitating inquiry into B. pseudomallei virulence.Burkholderia pseudomallei is a betaproteobacterium and causative agent of melioidosis in humans. B. pseudomallei infections are most common in Southeast Asia and Northern Australia, where melioidosis is endemic (13), and typically result from contact with contaminated soil and water after wounding or inhalation (12,13,33,65). Disease onset can be rapid or sometimes after a latency of years (42, 46), with clinical manifestations ranging from localized dermal lesions and soft tissue abscesses to acute septicemia and pneumonia. Due to its prevalence in soils in areas of Thailand, high infectivity, and the fact that melioidosis can be fatal even with antibiotic therapy (13), B. pseudomallei is considered a biothreat agent and as such is designated a category B select agent restricted to biosafety level 3 (BSL-3) by the Centers for Disease Control and Prevention (53).Despite its medical significance and biosecurity implications, relatively few molecular details exist regarding factors contributing to the heightened infectivity and lethality of B. pseudomallei relative to other bacterial pathogens. The majority of validated B. pseudomallei virulence factors are those which are conserved in other pathogens, e.g., exopolysaccharide capsule (3, 16, 51), lip...

Dissection of Francisella-Host Cell Interactions in Dictyostelium discoideum

“…5A and B) (14,17,21,33). As with other pathogenic bacteria (54,(83)(84)(85)(86), Dictyostelium was not able to feed on a lawn of F. noatunensis subsp. noatunensis.…”

Melioidosis: molecular aspects of pathogenesis

“…The BPSL0127-0128 TCS was identified using the phagocytic amoeba Dictyostelium discoideum as a model system. Mutation of the sensor kinase (BPSL0127) in B. pseudomallei resulted in decreased resistance to D. discoideum predation, reduced survival in RAW 264.7 cells, and was avirulent in BALB/c mice [112]. The specific genes controlled by BPSL0127-0128 remain to be determined.…”

Cross-Species Comparison of the Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei Quorum-Sensing Regulons

“…Though B. thailandensis is considered nonpathogenic, it is able to replicate in cultured mammalian cells and is able to resist predation by amoeba (45,46). B. thailandensis and B. pseudomallei mutants in this ortholog (BP1026B_II0970/BTH_II1513) showed reduced survival in Dictyostelium discoideum amoeba but were able to replicate to wild type-levels during intracellular infection assays in RAW 264.7 cells (46). In Burkholderia cenocepacia, an orthologous gene acts as an enhancer of AHL-mediated phenotypes (47).…”