Molecular and cellular characterisation of Burkholderia pseudomallei interactions with host cells

Abstract

Burkholderia pseudomallei is a bacterial pathogen that causes a broad spectrum of serious and oftenfatal diseases, collectively termed melioidosis. B. pseudomallei is capable of interacting specifically with host cells; it can adhere, invade, and survive and replicate intracellularly and induce the formation of multinucleated giant cells (MNGCs). While recent work has begun characterising these interactions at the molecular level, much is still unknown. In this work, B. pseudomallei interactions with eukaryotic cells were investigated at the cellular and molecular level. Many putative loci were studied by, firstly, generating isogenic mutant strains lacking a gene or locus of interest; and secondly, by characterising each mutant strain using a number of in vitro and in vivo models. These included models for adherence to eukaryotic cells, microcolony development, biofilm formation, twitching motility, bacterial cell aggregation, invasion of eukaryotic cells, actinbased motility, intracellular proliferation, multinucleated giant cell (MNGC) development and characterisation, gene expression, and virulence in vivo. Three putative type IV pilus (TFP) systems were identified in nine loci in the B. pseudomallei K96243 genome; type IVA, IVB, and IVB Flp pilus systems. Studies revealed that TFP are produced on the surface of B. pseudomallei K96243 but that TFP play only a minor role in adherence to eukaryotic cells using the assays described. However, collaborators showed that B. pseudomallei K96243 uses the type IVA pilin, PilA, to adhere to eukaryotic cells using a different adherence assay. Interestingly, two strains of B. pseudomallei adhered to eukaryotic cells to different degrees and used TFP (pilA) differently. B. pseudomallei K96243 adhered significantly less to four cell lines than B. pseudomallei 08. Furthermore, adherent microcolony formation was shown to be a temperaturedependent phenotype that enhanced bacterial association with eukaryotic cells; however, while B. pseudomallei 08 required pilA to form microcolonies, strain K96243 did not form microcolonies, and cell association was markedly reduced for K96243 relative to strain 08. TFP were not required for the formation of biofilms on PVC; however, type IVA and IVB Flp pili were required for optimal virulence in BALB/c mice using the intranasal route of infection, indicating the importance of TFP for B. pseudomallei survival in vivo. This work also identified that growth temperature, growth medium, and association with eukaryotic cells were important regulatory signals for adherence/cell association, microcolony formation, biofilm development, and TFP (pilA) expression. A homologue of eukaryotic 'senescence marker protein30' (SMP30) was identified in B. pseudomallei 08 and K96243 and was termed Lfp1 (for lactonase family protein 1). lfp1 is located within a genomic island and is conserved in both prokaryotes and eukaryotes. A homologue of lfp1 (lfp2) was also present in B. pseudomallei K96243. Though sharing considerable homology, prokaryotic and eukaryotic homologues of lfp1 were shown to be phylogenetically distinct. Expression of lfp1 mRNA by B. pseudomallei 08 was significantly increased in association with eukaryotic cells, relative to maintenance media alone; however, lfp1 was not required for adherence, invasion, intracellular proliferation, actinbased motility, or cell fusion by B. pseudomallei. Importantly though, B. pseudomallei 08infected macrophagelike cells rapidly fused into MNGCs, assuming an lfp1specific osteoclastlike pattern of gene expression that was distinct from B. thailandensisinfected MNGCs, and the process may be different to the LPSdependent mechanisms of osteoclastlike cell induction described previously for other bacteria. B. pseudomalleiinduced MNGC formation correlated with potent increases in mRNA levels for the cell fusion and multinucleation factors MCP1, CCL9/MIP1?, RANTES, and NFATc1 in B. pseudomalleiinfected cells, implicating these molecules in cell fusion. B. pseudomalleiinduced osteoclastlike MNGCs could not authentically resorb dentine; however, regions of apparently demineralised dentine were observed, suggesting a defect in the excavation of the organic phase of bone, analogous to that observed in CTSK knockout mice. Finally, an lfp1 null mutant was significantly attenuated for virulence in both the Syrian hamster model and the BALB/c inhalation model, indicating a role for lfp1 in virulence.