Protective host immune responses to anthrax infection in humans and animal models are characterized by the development of neutralizing Abs against the receptor-binding anthrax protective Ag (PA), which, together with the lethal factor (LF) protease, composes anthrax lethal toxin (LT). We now report that B cells, in turn, are targets for LT. Anthrax PA directly binds primary B cells, resulting in the LF-dependent cleavage of the MAPK kinases (MAPKKs) and disrupted signaling to downstream MAPK targets. Although not directly lethal to B cells, anthrax LT treatment causes severe B cell dysfunction, greatly reducing proliferative responses to IL-4-, anti-IgM-, and/or anti-CD40 stimulation. Moreover, B cells treated with anthrax LT in vitro or isolated from mice treated with anthrax LT in vivo have a markedly diminished capacity to proliferate and produce IgM in response to TLR-2 and TLR-4 ligands. The suppressive effects of anthrax LT on B cell function occur at picomolar concentrations in vitro and at sublethal doses in vivo. These results indicate that anthrax LT directly inhibits the function of B cells in vitro and in vivo, revealing a potential mechanism through which the pathogen could bypass protective immune responses.

The human folate receptors (hFRs) are important in the cellular accumulation of folates and antifolates. We described the structure of the human KB cell FR (hFR-KB) gene and identified two discrete promoter regions (P1 and P4) upstream from exons 1 and 4, respectively (Elwood et al., 1993). To further understand the molecular basis of hFR expression, we have now analyzed the basal transcription of the P4 promoter localized upstream of a major transcription start site. The sequence upstream from exon 4 contains several potential transcriptional factor-binding sites and a consensus initiator region sequence at the transcription start site but does not contain canonical TATA or CAAT boxes. While deletion of a 5' flanking sequence from nt -1023 to nt -605 of P4 promoter region decreases the luciferase reporter gene expression in KB cells to 54-70% of control construct, the removal of the sequence between nt -292 and nt -46 markedly decreases the activity to 3%. DNase I footprints and competitive mobility shift and supershift mobility assays indicate that Sp1 or Sp1-related nuclear protein(s) bind to three clustered GC-rich regions within the sequence between nt -292 and nt -46 of the hFR-KB P4 promoter. Both in vitro and in vivo analyses of the expression of promoter constructs containing site-specific mutation(s) of these three Sp1-binding sites and initiator sequence demonstrate that each of three Sp1 sites and the initiator sequence are required for optimum promoter activity and that they interact cooperatively in this P4 promoter of the hFR-KB gene.