Burkholderia pseudomallei, a facultative intracellular bacterium, is the causative agent of a broad spectrum of diseases collectively known as melioidosis. Its ability to survive inside phagocytic and nonphagocytic cells and to induce multinucleated giant cell (MNGC) formation has been demonstrated. This study was designed to assess a possible mechanism(s) leading to this cellular change, using virulent and nonvirulent strains of B. pseudomallei to infect both phagocytic and nonphagocytic cell lines. We demonstrated that when the cells were labeled with two different cell markers (CMFDA or CMTMR), mixed, and then infected with B. pseudomallei, direct cell-to-cell fusion could be observed, leading to MNGC formation. Staining of the infected cells with rhodamine-conjugated phalloidin indicated that immediately after the infection, actin rearrangement into a comet tail appearance occurred, similar to that described earlier for other bacteria. The latter rearrangement led to the formation of bacterium-containing, actin-associated membrane protrusions which could lead to a direct cell-to-cell spreading of B. pseudomallei in the infected hosts. Results from 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) nuclear staining, poly-ADP ribose polymerase cleavage, staining of infected cells for phosphatidylserine exposure with annexin V, and electrophoresis of the DNA extracted from these infected cells showed that B. pseudomallei could kill the host cells by inducing apoptosis in both phagocytic and nonphagocytic cells.

MATERIALS AND METHODSImmunogens and Immunization Schedules. 10-to 12-week old female BALB/cAnN mice were injected with purified myelomaproteins 315 or 460, which were isolated by subjection of the proteins to mild reduction and alkylation (in this work with iodoacetic acid) (2, 3). Proteins 315 and 460 purified in this manner (820w,, = 6.5) have about eight carboxymethyl groups per molecule (150,000 daltons), one on the C-terminal or penultimate cysteine of the light chain and 3-4 on the heavy chain (4). Both purified proteins had intact ligandbinding sites, as shown by specific quenching of their tryptoAbbreviation: TD5o, number of cells required to produce tumors in 50% of control (untreated) animals; Dnp, 2,4-dinitrophenyl.

Burkholderia pseudomallei is a causative agent of melioidosis, a life threatening disease which affects humans and animals in tropical and subtropical areas. This bacterium is known to survive and multiply inside cells such as macrophages. The mechanism of host defense against this bacterium is still unknown.In this study, we demonstrated that B. pseudomallei exhibited unique macrophage activation activity compared with Escherichia coli and Salmonella typhi, The mouse macrophage cell line (RAW 264.7) infected with B. pseudomallei at MOl of 0.1:1, 1:1 and 10:1 did not express a detectable level of inducible nitric oxide synthase (iNOS). Moreover, the B. pseudomallei infected cells released TNF-a only when they were infected with high MOl (10:1). Unlike the cells infected with B. pseudomallei, the cells infected with E. coli, and S. typhi expressed iNOS even at MOl of 0.1:1. These infected cells also released a significantly higher level of TNF-a at the low MOl ratio. The cells that were preactivated with IFN-oy prior to being infected with B. pseudomallei exhibited an enhanced production of iNOS and TNF-a release. The increased macrophage activation activity in the presence of IFN -oy also correlated with the restriction of the intracellular bacteria survival. Moreover, IFN-oy also prevented cell fusion and multinucleated cell formation induced by B. pseudomallei, a phenomenon recently described by our group. Altogether, these results indicate that internalization of B. pseudomallei failed to trigger substantial macrophage activation, a phenomenon which could prolong their survival inside the phagocytic cells and facilitate a direct cell to cell spreading of B. pseudomallei to neighboring cells.

There is worldwide concern that the avian influenza H5N1 virus, with a mortality rate of >50%, might cause the next influenza pandemic. Unlike most other influenza infections, H5N1 infection causes a systemic disease. The underlying mechanisms for this effect are still unclear. In this study, we investigate the interplay between avian influenza H5N1 and human dendritic cells (DC). We showed that H5N1 virus can infect and replicate in monocyte-derived and blood myeloid DC, leading to cell death. These results suggest that H5N1 escapes viral-specific immunity, and could disseminate via DC. In contrast, blood pDC were resistant to infection and produced high amounts of IFN-α. Addition of this cytokine to monocyte-derived DC or pretreatment with TLR ligands protected against infection and the cytopathic effects of H5N1 virus.

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