Burkholderia pseudomallei Capsule Exacerbates Respiratory Melioidosis but Does Not Afford Protection against Antimicrobial Signaling or Bacterial Killing in Human Olfactory Ensheathing Cells.

Melioidosis, caused by the bacterium Burkholderia pseudomallei, is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.

Quorum sensing negatively regulates multinucleate cell formation during intracellular growth of Burkholderia pseudomallei in macrophage-like cells.

Burkholderia pseudomallei is a Gram-negative environmental bacterium and the causative agent of melioidosis, a potentially fatal, acute or chronic disease endemic in the tropics. Acyl homoserine lactone (AHL)-mediated quorum sensing and signalling have been associated with virulence and biofilm formation in numerous bacterial pathogens. In the canonical acyl-homoserine lactone signalling paradigm, AHLs are detected by a response regulator. B. pseudomallei encodes three AHL synthases, encoded by bpsI1, bpsI2 and bpsI3, and five regulator genes. In this study, we mutated the B. pseudomallei AHL synthases individually and in double and triple combination. Five AHLs were detected and quantified by tandem liquid chromatography-mass spectroscopy. The major AHLs produced were N-octanoylhomoserine lactone and N-(3-hydroxy-decanoyl)homoserine lactone, the expression of which depended on bpsI1 and bpsI2, respectively. B. pseudomallei infection of macrophage cells causes cell fusion, leading to multinucleated cells (3 or more nuclei per cell). A triple mutant defective in production of all three AHL synthases was associated with a striking phenotype of massively enhanced host cellular fusion in macrophages. However, neither abrogation of host cell fusion, achieved by mutation of bimA or hcp1, nor enhancement of fusion altered intracellular replication of B. pseudomallei. Furthermore, when tested in murine models of acute melioidosis the AHL synthase mutants were not attenuated for virulence. Collectively, this study identifies important new aspects of the genetic basis of AHL synthesis in B. pseudomallei and the roles of these AHLs in systemic infection and in cell fusion in macrophages for this important human pathogen.