The Brucella abortus virB operon, encoding a type IV secretion system (T4SS), is required for intracellular replication and persistent infection in the mouse model. The products of the first two genes of the virB operon, virB1 and virB2, are predicted to be localized at the bacterial surface, where they could potentially interact with host cells. Studies to date have focused on characterization of transposon mutations in these genes, which are expected to exert polar effects on downstream genes in the operon. In order to determine whether VirB1 and VirB2 are required for the function of the T4SS apparatus, we constructed and characterized nonpolar deletion mutations of virB1 and virB2. Both mutants were shown to be nonpolar, as demonstrated by their ability to express the downstream gene virB5 during stationary phase of growth in vitro. Both VirB1 and VirB2 were essential for intracellular replication in J774 macrophages. The nonpolar virB2 mutant was unable to cause persistent infection in the mouse model, demonstrating the essential role of VirB2 in the function of the T4SS apparatus during infection. In contrast, the nonpolar virB1 mutant persisted at wild-type levels, showing that the function of VirB1 is dispensable in the mouse model of persistent infection.Brucella spp. are found in association with a large number of wild and domesticated animal species, where they can cause persistent infection and abortion. Zoonotic transmission of the organism to humans can lead to a chronic febrile disease known as brucellosis or Malta fever. A characteristic of both natural and zoonotic forms of the disease is extended survival of the organism in tissues of the reticuloendothelial system, such as the spleen, lymph nodes, and bone marrow.In a mutant screen for Brucella abortus virulence factors required for survival in the murine reticuloendothelial system, the virB operon, encoding homologs of Agrobacterium tumefaciens and Bordetella pertussis type IV secretion systems (24), was found to be essential for persistence in mice (18). The importance of these genes for intracellular survival was demonstrated in tissue culture models of infection as well (12,15,20,24,30). These genes were subsequently found to be expressed intracellularly by Brucella suis in macrophages (8), where they are required for localization of B. abortus to an intracellular niche that is associated with the endoplasmic reticulum (9), the same location where B. abortus was earlier shown to replicate in nonprofessional phagocytes (13,14,27) and in the ruminant placenta (2, 23). For B. abortus, the virB genes have been implicated in the initial interactions between the bacterium and the host cell during entry into macrophages (33).Based on studies performed with A. tumefaciens, both VirB1 and VirB2 are predicted to be accessible on the surface of B. abortus. VirB2 is predicted to form a pilus-like structure, and VirB1 is predicted to have two domains-a lytic transglycosylase and a second domain that is released to the extracellular medium and remains a...

CRH-binding protein (CRH-BP) regulates activation of the hypothalamic-pituitary-adrenal (HPA) axis by binding and inhibiting CRH. We investigated for the first time transcriptional regulation of the human CRH-BP promoter using transient transfections. Estrogen receptors (ERs) contributed to ligand-independent constitutive activation of the promoter, whereas in the presence of estradiol ERalpha induced and ERbeta repressed promoter activity in a dose-dependent manner. TNFalpha inhibited promoter induction by ERalpha in the absence and presence of estradiol. Three ERE half-sites in the CRH-BP promoter bound ERalpha and ERbeta in an EMSA, and disruption of ERE half-sites by site-directed mutagenesis abolished ligand-independent induction by ERalpha and ERbeta and promoter enhancement by estradiol-activated ERalpha. Repression by estradiol/ERbeta was unaffected by disruption of ERE half-sites, activating protein 1, cAMP response element, GATA, or nuclear factor kappaB sites, and reversed to promoter induction by estrogen antagonists, tamoxifen and ICI 182,780, suggesting corepressor involvement. In hypothalamic GT1-7 cells, Western blotting demonstrated rapid induction of endogenous CRH-BP expression by estradiol-bound ER, which was inhibited by TNFalpha. We propose a model in which ERs maintain basal CRH-BP expression in pituitary and neurosecretory cells, whereas in the presence of ERalpha estrogen enhances CRH-BP transcription, causing down-regulation of the HPA axis, and nuclear factor kappaB-activating cytokines activate the HPA axis by inhibiting ERalpha.

Mucosal pentraxin (Mptx), identified in rats, is a short pentraxin of unknown function. Other subfamily members are Serum amyloid P component (SAP), Creactive protein (CRP) and Jeltraxin. Rat Mptx mRNA is predominantly expressed in colon and in vivo is strongly (30-fold) regulated by dietary heme and calcium, modulators of colon cancer risk. This renders Mptx a potential nutrient sensitive biomarker of gut health. To support a role as biomarker, we examined whether the pentraxin protein structure is conserved, whether Mptx protein is nutrientsensitively expressed and whether Mptx is expressed in mouse and human. Sequence comparison and 3D modelling showed that rat Mptx is highly homologous to the other pentraxins. The calcium-binding site and subunit interaction sites are highly conserved, while a loop deletion and charged residues contribute to a distinctive ''top'' face of the pentamer. In accordance with mRNA expression, Mptx protein is strongly down-regulated in rat colon mucosa in response to high dietary heme intake. Mptx mRNA is expressed in rat and mouse colon, but not in human colon. A stop codon at the beginning of human exon two indicates loss of function, which may be related to differences in intestinal cell turnover between man and rodents.