Multinucleated Giant Cell Formation and Apoptosis in Infected Host Cells Is Mediated by Burkholderia pseudomallei Type III Secretion Protein BipB

Abstract

Here we have assessed the role of a type III translocator protein, BipB, in the cell biology and virulence of Burkholderia pseudomallei. Genetic inactivation of bipB reduced multinucleated giant cell formation, cell-to-cell spreading of bacteria, and induction of apoptosis of J774A.1 macrophages. The bipB mutant was also significantly attenuated following intranasal challenge of BALB/c mice, whereas virulence was fully restored by complementation with a functional bipB gene. Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, is a gram-negative bacterium. Melioidosis is endemic in southeast Asia and tropical Australia and has been reported sporadically elsewhere (6). Currently, there is no vaccine against melioidosis. Uniquely among intracellular bacterial pathogens, B. pseudomallei induces host cell fusion leading to multinucleated giant cell (MNGC) formation in tissue culture models of infection (14). This novel phenotype may be relevant to pathogenesis, since granuloma formation and generation of MNGC are also found in tissues of humans with melioidosis (23). In addition to inducing MNGC formation, B. pseudomallei is able to spread from cell to cell and induce apoptotic death in infected host cells (14). The molecular mechanisms of these pathogenic characteristics have not been elucidated. Analysis of the B. pseudomallei genome and several other studies have demonstrated the presence of a type III secretion system (TTSS) (for reviews, see references 3, 12, 17, 20, and 22). A knockout mutant of B. pseudomallei lacking a functional bipD gene, a homologue of Salmonella enterica serovar Typhimurium sipD, on the TTSS3/bsa cluster of TTSS exhibited reduced replication in murine macrophage-like cells (20), was significantly attenuated in BALB/c mice and gave partial protection against subsequent challenge with wild-type B. pseudomallei (19). These data correlated with the recent report that the TTSS3/bsa cluster is required for the pathogenicity of B. pseudomallei (21). In addition to BipD, B. pseudomallei BipB and BipC (46 and 30% amino acid identity to Salmonella SipB and SipC, respectively) have been identified in the TTSS3/bsa cluster (3). Here, we report on the role of BipB in the pathogenesis of infection with B. pseudomallei. With Salmonella organisms, purified SipB integrates into artificial membranes and induces liposome fusion (10), and it is required for inducing apoptosis in murine macrophages (11). By analogy with SipB, therefore, we investigated the role of BipB for MNGC formation, cell-to-cell spreading, and induction of apoptosis in infected host cells. We also examined the virulence of a B. pseudomallei bipB mutant in a murine model of melioidosis.