The bacterial gene lfpA influences the potent induction of calcitonin receptor and osteoclast-related genes in Burkholderia pseudomallei-induced TRAP-positive multinucleated giant cells

Abstract

SummaryBurkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis, a spectrum of potentially fatal diseases endemic in Northern Australia and South-East Asia. We demonstrate that B. pseudomallei rapidly modifies infected macrophage-like cells in a manner analagous to osteoclastogenesis. These alterations include multinucleation and the expression by infected cells of mRNA for factors required for osteoclastogenesis: the chemokines monocyte chemotactic protein 1 (MCP-1), macrophage inflammatory protein 1 gamma (MIP-1g), 'regulated on activation normal T cell expressed and secreted' (RANTES) and the transcription factor 'nuclear factor of activated T-cells cytoplasmic 1' (NFATc1). An increase in expression of these factors was also observed after infection with Burkholderia thailandensis. Expression of genes for the osteoclast markers calcitonin receptor (CTR), cathepsin K (CTSK) and tartrate-resistant acid phosphatase (TRAP) was also increased by B. pseudomallei-infected, but not by B. thailandensisinfected cells. The expression by B. pseudomalleiinfected cells of these chemokine and osteoclast marker genes was remarkably similar to cells treated with RANKL, a stimulator of osteoclastogenesis.Analysis of dentine resorption by B. pseudomalleiinduced osteoclast-like cells revealed that demineralization may occur but that authentic excavation does not take place under the tested conditions. Furthermore, we identified and characterized lfpA (for lactonase family protein A) in B. pseudomallei, which shares significant sequence similarity with the eukaryotic protein 'regucalcin', also known as 'senescence marker protein-30' (SMP-30). LfpA orthologues are widespread in prokaryotes and are well conserved, but are phylogenetically distinct from eukaryotic regucalcin orthologues. We demonstrate that lfpA mRNA expression is dramatically increased in association with macrophage-like cells. Mutation of lfpA significantly reduced expression of the tested host genes, relative to the response to wild-type B. pseudomallei. We also show that lfpA is required for optimal virulence in vivo.

Dissection of the Burkholderia intracellular life cycle using a photothermal nanoblade

“…In considering how these and earlier observations might be related, a closer look at the process of plaque formation itself is revealing. Bp, Bm, and Bt induce cell fusion and multinucleate giant cell (MNGC) formation with remarkable efficiency in a variety of cell types (2,10,25). In observing cell monolayers following nanoblade delivery, we often noticed the appearance of MNGCs in areas that developed to become plaques.…”

Burkholderia mallei Cluster 1 Type VI Secretion Mutants Exhibit Growth and Actin Polymerization Defects in RAW 264.7 Murine Macrophages

“…this process (7,24,28). To date, both BipB and RpoS have been implicated in B. pseudomallei-induced MNGC formation in murine macrophage cell lines (54,58).…”

“…mallei and B. pseudomallei are known to cause MNGC formation both in vitro and in vivo (7,11,20,24,28,49). At present, the relevance of MNGCs with respect to virulence is unclear; however, it has been proposed that giant cells may provide these pathogens with an immune-privileged niche in which to survive and replicate within a host (7). The formation MNGCs from mononuclear cells is thought to be a result of cell fusion events; however, very little is known about the bacterial and host cell factors that are involved in 96 BURTNICK ET AL.…”

Interaction of Burkholderia pseudomallei and Burkholderia thailandensis with human monocyte-derived dendritic cells

“…However, we have previously shown that an acapsulate B. pseudomallei mutant is capable of infecting nasal-associated lymphoid tissue and, presumably, DCs also (Owen et al, 2009). Similarly, acapsulate B. thailandensis is capable of invasion and replication in murine phagocytic cells, albeit with an altered host-cell response (Boddey et al, 2007), and the capacity of B. thailandensis to induce lung damage, suggested to be mediated, in part, by inflammatory cytokine production, have been reported (West et al, 2008). In the present study, we assessed whether the presence of capsular polysaccharide affects bacterial survival and found that neither the acapsular mutant of B. pseudomallei nor a strain of B. thailandensis differed from capsulate B. pseudomallei in their interactions with MDDCs.…”

“…Meanwhile, imipenem is lipid insoluble and penetrates cells poorly if at all (Menashe et al, 2008;Murdoch & Peterson, 1991;Sahly et al, 2008). Numerous other studies have used imipenem (Boddey et al, 2007;Brown et al, 2004;MacDonald & Speert, 2008) rather than kanamycin. We have compared the effects of Table 1.…”