Because of Burkholderia pseudomallei's classification as a select agent in the United States, genetic manipulation of this bacterium is strictly regulated. Only a few antibiotic selection markers, including gentamicin, kanamycin, and zeocin, are currently approved for use with this bacterium, but wild-type strains are highly resistant to these antibiotics. To facilitate routine genetic manipulations of wild-type strains, several new tools were developed. A temperature-sensitive pRO1600 broad-host-range replicon was isolated and used to construct curable plasmids where the Flp and Cre recombinase genes are expressed from the rhamnose-regulated Escherichia coli P BAD promoter and kanamycin (nptI) and zeocin (ble) selection markers from the constitutive Burkholderia thailandensis ribosomal P S12 or synthetic bacterial P EM7 promoter. Flp and Cre site-specific recombination systems allow in vivo excision and recycling of nptII and ble selection markers contained on FRT or loxP cassettes. Finally, expression of Tn7 site-specific transposase from the constitutive P1 integron promoter allowed development of an efficient site-specific chromosomal integration system for B. pseudomallei. In conjunction with a natural transformation method, the utility of these new tools was demonstrated by isolating an unmarked ⌬(amrRAB-oprA) efflux pump mutant. Exploiting natural transformation, chromosomal DNA fragments carrying this mutation marked with zeocin resistance were transferred between the genomes of two different B. pseudomallei strains. Lastly, the deletion mutation was complemented by a chromosomally integrated mini-Tn7 element carrying the amrAB-oprA operon. The new tools allow routine select-agentcompliant genetic manipulations of B. pseudomallei and other Burkholderia species.

These data suggest that the olfactory nerve is the route of entry into the brain and that this route of entry may be paralleled in cases of human neurologic melioidosis. This study focuses attention on the upper respiratory tract as a portal of entry, specifically focusing on NALT as a route for the development of systemic infection via the bloodstream and on the olfactory epithelium as a direct route to the brain.

Successful pregnancy in humans has been associated with production of IL-4 by T cells at the feto–maternal interface. Soluble HLA-G5 produced by trophoblasts potentially controls the decidual T cell cytokine profile. We studied the effect of HLA-G5 on the cytokine profile of purified human macrophages and Ag-specific T cells in vitro. We demonstrated that HLA-G5 increased production of IL-12 by purified peripheral blood macrophages. Although IL-12 production by macrophages is known to induce IFN-γ production by CD4+ T cells, HLA-G5 increased production of IL-4 but not IFN-γ by CD4+ T cells after Ag presentation by macrophages. We found that this apparent paradox was due to the differential expression of the ILT2 HLA-G5 receptor on activated T cells and macrophages. This receptor was upregulated in the former and downregulated in the latter after Ag presentation and activation of both cell types. This observation was confirmed in situ, where decidual macrophages and T cells are continuously exposed to HLA-G5 produced locally and activated by trophoblast alloantigens. Freshly isolated decidua basalis macrophages expressed lower levels of ILT2 than peripheral blood macrophages from the same pregnant women. They did not spontaneously produce IL-12, whereas freshly isolated decidual CD4+ T cells expressed high levels of activation markers (CD25, HLA-DR, and CD69) as well as ILT2 and spontaneously produced IL-4 but not IFN-γ. Therefore, HLA-G5 could be responsible, at least in part, via its interaction with ILT2, for decidual T cell IL-4 production, known to be crucial for successful pregnancy.