IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice.

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

Analysis of catabolite control protein A-dependent repression in Staphylococcus xylosus by a genomic reporter gene system.

A single-copy reporter system for Staphylococcus xylosus has been developed, that uses a promoterless version of the endogenous beta-galactosidase gene lacH as a reporter gene and that allows integration of promoters cloned in front of lacH into the lactose utilization gene cluster by homologous recombination. The system was applied to analyze carbon catabolite repression of S. xylosus promoters by the catabolite control protein CcpA. To test if lacH is a suitable reporter gene, beta-galactosidase activities directed by two promoters known to be subject to CcpA regulation were measured. In these experiments, repression of the malRA maltose utilization operon promoter and autoregulation of the ccpA promoters were confirmed, proving the applicability of the system. Subsequently, putative CcpA operators, termed catabolite-responsive elements (cres), from promoter regions of several S. xylosus genes were tested for their ability to confer CcpA regulation upon a constitutive promoter, P(vegII). For that purpose, cre sequences were placed at position +3 or +4 within the transcribed region of P(vegII). Measurements of beta-galactosidase activities in the presence or absence of glucose yielded repression ratios between two- and eightfold. Inactivation of ccpA completely abolished glucose-dependent regulation. Therefore, the tested cres functioned as operator sites for CcpA. With promoters exclusively regulated by CcpA, signal transduction leading to CcpA activation in S. xylosus was examined. Glucose-dependent regulation was measured in a set of isogenic mutants showing defects in genes encoding glucose kinase GlkA, glucose uptake protein GlcU, and HPr kinase HPrK. GlkA and GlcU deficiency diminished glucose-dependent CcpA-mediated repression, but loss of HPr kinase activity abolished regulation. These results clearly show that HPr kinase provides the essential signal to activate CcpA in S. xylosus. Glucose uptake protein GlcU and glucose kinase GlkA participate in activation, but they are not able to trigger CcpA-mediated regulation independently from HPr kinase.

Eradication of disseminated lymphomas with CpG-DNA activated T helper type 1 cells from nontransgenic mice.

Various evidence suggests that adoptive transfer of polyclonal, tumor-specific, IFN-gamma-producing CD4+ T cells [T helper type 1 (Th1) cells] should be highly efficient for tumor immune therapy. However, this approach could not be tested because very few MHC class II-restricted tumor peptides have been defined. Here we show that stimulation of freshly isolated T helper cells with syngeneic tumor cells and antigen-presenting cells in the presence of immunostimulatory CpG DNA allows the generation of large numbers of strongly polarized, tumor-specific Th1 cells within 3 weeks of culture, even when T helper cells were derived from tumor-bearing mice. A single injection of 0.5 x 10(6) A20-specific Th1 cells even eradicated disseminated A20 lymphomas and provided lifelong protection without inducing autoimmune disease. The therapy was largely independent of CD8+ cells but required IFN-gamma and CD40-CD40L interactions, suggesting that tumor-specific Th1 cells eradicate established tumors by activating proinflammatory macrophages.

CpG oligodeoxynucleotides trigger protective and curative Th1 responses in lethal murine leishmaniasis.

Synthetic oligodeoxynucleotides containing CpG dinucleotides (CpG-ODN) mimic the immunostimulatory qualities of bacterial DNA. We asked whether immunostimulation by CpG-ODN predisposes for a commitment toward a Th1 vs a Th2 response in Leishmania major infection, a model for a lethal Th2-driven disease, in BALB/c mice. CpG-ODN induced Th1 effector T cells in vitro and conveyed protective immunity to disease-prone BALB/c mice in vivo. Conversion to a Th1-driven resistant phenotype was associated with IL-12 production and maintained the expression of IL-12R beta2-chains. Most strikingly, CpG-ODN were even curative when given as late as 20 days after lethal L. major infection, indicating that CpG-ODN revert an established Th2 response. These findings imply an important role of bacterial DNA and CpG-ODN in the instruction of adaptive immune responses. They also point to the therapeutic potential of CpG-ODN in redirecting curative Th1 responses in Th2-driven disorders.

Catabolite repression mediated by the catabolite control protein CcpA in Staphylococcus xylosus.

The gene ccpA encoding the catabolite control protein CcpA of Staphylococcus xylosus has been cloned and characterized. The CcpA protein belongs to the Lacl/GaiR family of bacterial regulators and is comprised of 329 amino acids, with a molecular mass of 36.3 kDa. It shows 56% identity with the CcpA proteins of Bacillus subtills and Bacillus megaterium. Inactivation of the ccpA gene in the genome of S. xylosus relieved the activities of three enzymes, alpha-glucosidase, beta-glucuronidase, and beta-galactosidase, from cataboilte repression by several carbohydrates. Concomitantly, transcription initiation of the maltose-utilization operon malRA, including the alpha-glucosidase gene malA, was no longer subject to glucose-specific control. Carbon source-dependent malRA regulation was also lost upon deletion of a palindromic sequence in the malRA promoter region resembling the catabolite-responsive elements essential for CcpA-dependent catabolite repression in Bacillus. These results strongly suggest that S. xylosus CcpA controls transcription of catabolite-repressible genes and operons by binding to catabolite-responsive operators when rapidly metabolizable carbohydrates are available. Accordingly, the cloned S. xylosus ccpA gene could complement the ccpA mutation in B. subtilis. The ccpA gene of S. xylosus is transcribed from two promoters, one of which is subject to autogenous repression by CcpA. Autoregulation results in a slight reduction of CcpA protein in glucose-grown cells. The characterization of the role of CcpA in carbon catabolite repression in S. xylosus demonstrates that a regulatory mechanism originally detected in Bacillus applies to another Gram-positive bacterium with low GC content.

Glucose kinase-dependent catabolite repression in Staphylococcus xylosus.

By transposon Tn917 mutagenesis, 16 mutants of Staphylococcus xylosus were isolated that showed higher levels of beta-galactosidase activity in the presence of glucose than the wild-type strain. The transposons were found to reside in three adjacent locations in the genome of S. xylosus. The nucleotide sequence of the chromosomal fragment affected by the Tn917 insertions yielded an open reading frame encoding a protein with a size of 328 amino acids with a high level of similarity to glucose kinase from Streptomyces coelicolor. Weaker similarity was also found to bacterial fructokinases and xylose repressors of gram-positive bacteria. The gene was designated glkA. Immediately downstream of glkA, two open reading frames were present whose deduced gene products showed no obvious similarity to known proteins. Measurements of catabolic enzyme activities in the mutant strains grown in the presence or absence of sugars established the pleiotropic nature of the mutations. Besides beta-galactosidase activity, which had been used to detect the mutants, six other tested enzymes were partially relieved from repression by glucose. Reduction of fructose-mediated catabolite repression was observed for some of the enzyme activities. Glucose transport and ATP-dependent phosphorylation of HPr, the phosphocarrier of the phosphoenolpyruvate:carbohydrate phosphotransferase system involved in catabolite repression in gram-positive bacteria, were not affected. The cloned glkA gene fully restored catabolite repression in the mutant strains in trans. Loss of GlkA function is thus responsible for the partial relief from catabolite repression. Glucose kinase activity in the mutants reached about 75% of the wild-type level, indicating the presence of another enzyme in S. xylosus. However, the cloned gene complemented an Escherichia coli strain in glucose kinase. Therefore, the glkA gene encodes a glucose kinase that participates in catabolite repression in S. xylosus.

Characterization of a genetic locus essential for maltose-maltotriose utilization in Staphylococcus xylosus.

A genetic locus from Staphylococcus xylosus involved in maltose-maltotriose utilization has been characterized. The chromosomal region was identified by screening a genomic library of S. xylosus in Escherichia coli for sucrose hydrolase activity. Nucleotide sequence analysis yielded two open reading frames (malR and malA) encoding proteins of 37.7 and 62.5 kDa, respectively. MalR was found to be homologous to the LacI-GalR family of transcriptional regulators, and MalA showed high similarity to yeast alpha-1,4-glucosidases and bacterial alpha-1,6-glucosidases. Inactivation of malA in the genome of S. xylosus led to a maltose-maltotriose-negative phenotype. In cell extracts of the mutant, virtually no glucose release from maltose and short maltodextrins was detectable. Inactivation of malA in a sucrose-6-phosphate hydrolase-deficient S. xylosus strain resulted in the complete loss of the residual sucrose hydrolase activity. The MalA enzyme has a clear preference for maltose but is also able to release glucose from short maltosaccharides. It cannot cleave isomaltose. Therefore, malA encodes an alpha-1,4-glucosidase or maltase, which also liberates glucose from sucrose. Subcloning experiments indicated that malA does not possess its own promoter and is cotranscribed with malR. Its expression could not be stimulated when maltose was added to the growth medium. Chromosomal inactivation of malR led to reduced maltose utilization, although alpha-glucosidase activity in the malR mutant was slightly higher than in the wild type. In the mutant strain, maltose uptake was reduced and inducibility of the transport activity was partially lost. It seems that MalR participates in the regulation of the gene(s) for maltose transport and is needed for their full expression. Thus, the malRA genes constitute an essential genetic locus for maltosaccharide utilization in S. xylosus