Like viruses, intracellular bacteria stimulate their host cells to produce type I IFNs (IFN-α and IFN-β). In our study, we investigated the signals and molecules relevant for the synthesis of and response to IFN by mouse macrophages infected with Listeria monocytogenes. We report that IFN-β is the critical immediate-early IFN made during infection, because the synthesis of all other type I IFN, expression of a subset of infection-induced genes, and the biological response to type I IFN was lost upon IFN-β deficiency. The induction of IFN-β mRNA and the IFN-β-dependent sensitization of macrophages to bacteria-induced death, in turn, was absolutely dependent upon the presence of the transcription factor IFN regulatory factor 3 (IRF3). IFN-β synthesis and signal transduction occurred in macrophages deficient for TLR or their adaptors MyD88, TRIF, or TRAM. Expression of Nod2, a candidate receptor for intracellular bacteria, increased during infection, but the protein was not required for Listeria-induced signal transduction to the Ifn-β gene. Based on our data, we propose that IRF3 is a convergence point for signals derived from structurally unrelated intracellular pathogens, and that L. monocytogenes stimulates a novel TLR- and Nod2-independent pathway to target IRF3 and the type I IFN genes.

No abstract

Four outer membrane proteins of Escherichia coli were examined for their capabilities and limitations in displaying heterologous peptide inserts on the bacterial cell surface. The T7 tag or multiple copies of the myc epitope were inserted into loops 4 and 5 of the ferrichrome and phage T5 receptor FhuA. Fluorescenceactivated cell sorting analysis showed that peptides of up to 250 amino acids were efficiently displayed on the surface of E. coli as inserts within FhuA. Strains expressing FhuA fusion proteins behaved similarly to those expressing wild-type FhuA, as judged by phage infection and colicin sensitivity. The vitamin B 12 and phage BF23 receptor BtuB could display peptide inserts of at least 86 amino acids containing the T7 tag. In contrast, the receptors of the phages K3 and , OmpA and LamB, accepted only insertions in their respective loop 4 of up to 40 amino acids containing the T7 tag. The insertion of larger fragments resulted in inefficient transport and/or assembly of OmpA and LamB fusion proteins into the outer membrane. Cells displaying a foreign peptide fused to any one of these outer membrane proteins were almost completely recovered by magnetic cell sorting from a large pool of cells expressing the relevant wild-type platform protein only. Thus, this approach offers a fast and simple screening procedure for cells displaying heterologous polypeptides. The combination of FhuA, along with with BtuB and LamB, should provide a comprehensive tool for displaying complex peptide libraries of various insert sizes on the surface of E. coli for diverse applications.The display of peptides on the surface of bacteria has become very attractive for a variety of applications such as the development of recombinant bacterial vaccines (32,33,34) and the screening of polypeptide libraries for protein-protein interactions (5, 27, 36). In Escherichia coli, the outer membrane proteins OmpA, LamB, and PhoE and also the flagellar and fimbrial proteins flagellin, FimH, and PapA (for a review, see reference 18) have been used to display peptides or proteins on the cell surface. However, insertion of peptides longer than 60 amino acids was shown to perturb the conformation of LamB and PhoE (1, 11), resulting in interference with proper cell surface localization. Similarly, the subunits of cellular appendages were also reported not to be suitable for the display of larger polypeptides (for a review, see reference 18). Although the lambda receptor is restricted for the size of insertion, it had been shown that a diverse variety of peptides could be displayed on the surface when fused to LamB (11). Subsequently, the adhesin AIDA-I (39) and the TraT protein (10) from Escherichia coli, as well as the ice-nucleation protein of Pseudomonas mobilis (26), were used to display heterologous polypeptides on the surface of E. coli. Whereas only peptide sequences of up to 100 amino acids were examined for display using the TraT protein, the AIDA-I and the ice-nucleation protein were shown to be capable of displaying a full-length pro...

Aberrant activation of the JAK-STAT pathway has been implicated in tumor formation; for example, constitutive activation of JAK2 kinase or the enforced expression of STAT5 induces leukemia in mice. We show here that the Janus kinase TYK2 serves an opposite function. Mice deficient in TYK2 developed Abelsoninduced B lymphoid leukemia/lymphoma as well as TEL-JAK2-induced T lymphoid leukemia with a higher incidence and shortened latency compared with WT controls. The cell-autonomous properties of Abelson murine leukemia virus-transformed (A-MuLV-transformed) TYK2 -/-cells were unaltered, but the high susceptibility of TYK2 -/-mice resulted from an impaired tumor surveillance, and accordingly, TYK2 -/-A-MuLV-induced lymphomas were easily rejected after transplantation into WT hosts. The increased rate of leukemia/lymphoma formation was linked to a decreased in vitro cytotoxic capacity of TYK2 -/-NK and NKT cells toward tumor-derived cells. RAG2/TYK2 double-knockout mice succumbed to A-MuLV-induced leukemia/lymphoma faster than RAG2 -/-TYK2 +/-mice. This defines NK cells as key players in tumor surveillance in Abelson-induced malignancies. Our observations provide compelling evidence that TYK2 is an important regulator of lymphoid tumor surveillance. IntroductionThe JAK-STAT pathway regulates cell proliferation, differentiation, and survival in hematopoietic cells (1, 2). Binding of a cytokine to its cognate receptor activates receptor-associated JAKs which in turn mediate the subsequent tyrosine phosphorylation of STAT proteins. Phosphorylated STAT proteins form dimers, translocate to the nucleus, and bind to specific DNA elements to induce or modulate expression of target genes. Gene knockout studies in mice underlined the vital role of the JAK-STAT pathway for hematopoiesis and other developmental processes (3).Recent interest had focused on the role of the JAK-STAT pathway in tumor formation. Aberrant activation of JAK-STAT signaling had been shown in multiple solid tumors and leukemia (4-9). In particular, STAT3 and STAT5 have drawn much attention; both transcription factors display oncogenic properties when expressed ectopically. A constitutively active mutant of STAT3 was shown to transform rat fibroblasts (10), and the enforced expression of WT STAT5 in the lymphoid lineage induced T cell leukemia in mice (11). In addition, JAK1 has been implicated in transformation by the v-abl as well as by the v-src oncogene (12, 13), and constitutive activation of JAK2 as in the TEL-JAK2 oncogene suffices to induce leukemia in mice and humans (14, 15). On the other hand, JAK1 was shown to be a tumor suppressor downstream of IFN-γ in a B lymphoid tumor model (16). A role as tumor suppressor downstream of IFN-γ has also been attributed to STAT1 (17-21). STAT1 -/-mice have been repeatedly used to study the role of IFN-γ in tumorigenesis and tumor surveillance (18,19). Recent evidence also proposed that STAT1 acted as a tumor suppressor by modulating apoptosis (22).TYK2 has been initially defined as a mediator of IFN-α/β sign...