Characterization of an Intracellular Flagellar System in Pathogenic Burkholderia Species
Burkholderia pseudomallei and Burkholderia thailandensis are related facultative intracellular pathogens that infect a variety of cell types, escape into the cytoplasm of host cells, and spread from cell-to-cell via type 6 secretion system (T6SS)-mediated cell fusion. Recent work has demonstrated a new mechanism of rapid intracellular motility dependent on a cryptic flagellar locus, fla2. In the first section of this dissertation, I show that the Fla2 system is present and expressed in Australian strains of B. pseudomallei and B. thailandensis. The results demonstrated that the fla2 locus encodes lateral flagella expressed intracellularly. This Fla2 system promotes swarming on semisolid agar. Lateral flagella encoded by the fla2 locus may provide a growth advantage for Burkholderia in environmental conditions, as wild-type B. thailandensis outcompeted a nonmotile ∆fliC2 derivative in a semisolid agar swarming competition assay.
In the next section of this dissertation I tested the effect of intracellular expression of lateral flagella on innate immune detection of Burkholderia. Innate immune mechanisms, particularly NLRC4-driven pyroptosis and production of IL-1β, are critical for the host response to B. pseudomallei infection. Such innate immune responses were previously attributed to type 3 secretion system (T3SS) stimuli deployed intracellularly. Here, I show that this response is at least partially dependent on fliC2, the flagellin gene encoded in the fla2 cluster in B. thailandensis and Australian strains of B. pseudomallei. While deletion of fliC2 in B. thailandensis had no measurable effect on pyroptosis or intracellular bacterial growth, it led to a nearly 33% decrease in IL-1β production in wild-type (wt) murine macrophages, without affecting IL-1β release in nlrc4-deficient macrophages. FliC2 did not appear to contribute similarly to IL-1β secretion during B. pseudomallei NAU14B-6 infection despite the immunogenicity of B. pseudomallei FliC2 ectopically expressed in a B. thailandensis ∆fliC2 strain. By quantitative RT-PCR, I demonstrate that intracellular expression of fliC2 is lower in the B. pseudomallei strains tested than in B. thailandensis. This difference may provide an explanation for the discrepancy between the innate immune response observed against FliC2 in B. thailandensis versus B. pseudomallei, leaving open the possibility that differences in fliC2 expression levels between B. pseudomallei strains may lead to strain-specific innate immune responses.
In the final section of this dissertation, I describe the role of a novel fla2 regulator on the global regulatory cascade involved in the B. pseudomallei intracellular life cycle. Overexpression of the flagellar response regulator frr activates the expression of lateral flagella, but decreases plaquing efficiency in both Fla2+ and Fla2- strains of B. pseudomallei and B. thailandensis. I show through quantitative RT-PCR that frr affects not only expression of lateral flagella components, but also polar flagella components from the Fla1 system utilized for extracellular swimming by Burkholderia and type 3 secretion system components required for escape from an initial endosome and from autophagosomes. These results suggest that frr plays a role as a master regulator controlling multiple aspects of Burkholderia virulence. In this chapter, I also investigate the role of other B. pseudomallei master regulators on subsets of virulence genes and demonstrate that each of these regulators exhibits crosstalk with other regulatory systems involved in the intracellular life cycle.
Together, the results described in this dissertation define and characterize a novel intracellular motility system in B. pseudomallei. The expression of lateral flagella by Burkholderia in the host cytoplasm is a unique and surprising feature for an intracellular pathogen, given the cytosolic surveillance mechanisms evolved in mammalian hosts to respond to flagella. In the concluding remarks of this dissertation, I discuss the status of B. pseudomallei as an “accidental” pathogen and how mechanisms evolved to survive and persist in specific ecological niches can contribute to or stand in the way of mammalian pathogenesis.