Loss of the LAT adaptor converts antigen-responsive T cells into pathogenic effectors that function independently of the T cell receptor.

Despite compromised T cell antigen receptor (TCR) signaling, mice in which tyrosine 136 of the adaptor linker for activation of T cells (LAT) was constitutively mutated (Lat(Y136F) mice) accumulate CD4(+) T cells that trigger autoimmunity and inflammation. Here we show that equipping postthymic CD4(+) T cells with LATY136F molecules or rendering them deficient in LAT molecules triggers a lymphoproliferative disorder dependent on prior TCR engagement. Therefore, such disorders required neither faulty thymic T cell maturation nor LATY136F molecules. Unexpectedly, in CD4(+) T cells recently deprived of LAT, the proximal triggering module of the TCR induced a spectrum of protein tyrosine phosphorylation that largely overlapped the one observed in the presence of LAT. The fact that such LAT-independent signals result in lymphoproliferative disorders with excessive cytokine production demonstrates that LAT constitutes a key negative regulator of the triggering module and of the LAT-independent branches of the TCR signaling cassette.

Repertoire of HLA-DR1-restricted CD4 T cell responses to capsular Caf1 antigen of Y. pestis HLA-transgenic mice

Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T cell responses for Caf1. We characterized CD4 T cell epitopes of Caf1 in 'humanized'-HLA-DR1 transgenic mice lacking endogenous MHC class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T cell immunity was measured with respect to proliferative and IFNgamma T cell responses and recognition by a panel of T cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased towards a single immunodominant epitope near the C-terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.

Use of antigen mimics to produce specific antibodies to anti-coccidial drugs

A range of polyclonal antibodies was successfully produced to the coccidiostatic drugs diclazuril and robenidine. Initial attempts to make immunogenic complexes of both drugs were ineffective due to difficulties encountered while trying to couple the compounds to large carrier proteins. Structural mimics, which could act as haptens for each drug, were sought and identified. The compounds identified were more open to chemical manipulation and were conjugated to carrier proteins to produce effective immunogens. The most sensitive antisera produced displayed IC(50)s of 1.5 ng/ml and 13 ng/ml for diclazuril and robenidine respectively. The antibody for diclazuril was shown to be specific, cross-reacting only with clazuril by 15%. The robenidine antibody displayed a low cross-reactivity of 1.2% to the compound used to produce the antibody. (C) 2007 Elsevier B.V. All rights reserved.

The Role of Direct Presentation by Donor Dendritic Cells in Rejection of Minor Histocompatibility Antigen-Mismatched Skin and Hematopoietic Cell Grafts

Background. The success of transplantation is hampered by rejection of the graft by alloreactive T cells. Donor dendritic cells (DC) have been shown to be required for direct priming of immune responses to antigens from major histocompatibility complex-mismatched grafts. However, for immune responses to major histocompatibility complex-matched, minor histocompatibility (H) antigen mismatched grafts, the magnitude of the T-cell response to directly presented antigens is reduced, and the indirect pathway is more important. Therefore, we aimed to investigate the requirement for donor DC to directly present antigen from minor H antigen mismatched skin and hematopoietic grafts.

T lymphocyte epitope mapping of porcine circovirus type 2

Immunoreactive T lymphocyte epitopes within the ORF1, ORF2, and ORF 3 products of porcine circovirus type 2 (PCV2) were mapped. For this, overlapping linear 20-mer peptides were synthesized and tested for their ability to induce T lymphocyte proliferation in porcine peripheral blood mononuclear cells (PBMCs) isolated from experimentally PCV2-infected pigs. After a preliminary screening of 31 (ORF1), 23 (ORF2), and 10 (ORF3) peptides using PBMCs from 4 PCV2-infected pigs, none of the peptides appeared to be immunoreactive (stimulation index [SI] : 2) in all four pigs. Only 14 peptides appeared to be immunoreactive in 3 of the 4 pigs. These peptides were designated as immunodominant in the preliminary screening and selected for further analysis. The immunodominant peptides were resynthesized and purified by high-performance liquid chromatography and tested for their ability to induce T lymphocyte proliferation in PBMCs from another three PCV2-infected pigs. None of the immunodominant peptides appeared to be immunoreactive in all three pigs of the second screening. Only three peptides appeared to be immunoreactive in two of three pigs, two encoded by PCV2 ORF1 (amino acid residues 81-100 and 201-220) and one encoded by PCV2 ORF3 (amino acid residues 31-50), and were therefore considered to be immunodominant in both screenings. Although peptides encoded by ORF2 appeared to show the highest immunoreactivity in some pigs, none of these peptides displayed immunodominance in both screenings. In summary, the present study indicates that the T lymphocyte responses to PCV2 are primarily directed toward epitopes of the nonstructural proteins of ORF1 and ORF3.

The WaaL O-antigen lipopolysaccharide ligase has features in common with metal ion-independent inverting glycosyltransferases(*)

WaaL is a membrane enzyme that catalyzes a key step in lipopolysaccharide (LPS) synthesis: the glycosidic bonding of a sugar at the proximal end of the undecaprenyl-diphosphate (Und-PP) O-antigen with a terminal sugar of the lipid A-core oligosaccharide (OS). Utilizing an in vitro assay, we demonstrate here that ligation with purified Escherichia coli WaaL occurs without adenosine-5'-triphosphate (ATP) and magnesium ions. Furthermore, E. coli and Pseudomonas aeruginosa WaaL proteins cannot catalyze ATP hydrolysis in vitro. We also show that a lysine substitution of the arginine (Arg)-215 residue renders an active protein, whereas WaaL mutants with alanine replacements in the periplasmic-exposed residues Arg-215, Arg-288 and histidine (His)-338 and also the membrane-embedded aspartic acid-389 are nonfunctional. An in silico approach, combining predicted topological information with the analysis of sequence conservation, confirms the importance of a positive charge at the small periplasmic loop of WaaL, since an Arg corresponding to Arg-215 was found at a similar position in all the WaaL homologs. Also, a universally conserved H[NSQ]X(9)GXX[GTY] motif spanning the C-terminal end of the predicted large periplasmic loop and the membrane boundary of the transmembrane helix was identified. The His residue in this motif corresponds to His-338. A survey of LPS structures in which the linkage between O-antigen and lipid A-core OS was elucidated reveals that it is always in the beta-configuration, whereas the sugars bound to Und-PP are in the alpha-configuration. Together, our biochemical and in silico data argue that WaaL proteins use a common reaction mechanism and share features of metal ion-independent inverting glycosyltransferases.

Common Themes in Glycoconjugate Assembly Using the Biogenesis of O-Antigen Lipopolysaccharide as a Model System

The biosynthesis of glycoconjugates is remarkably conserved in all types of cells since the biochemical reactions involved exhibit similar characteristics, which can be summarized as follows: (a) the saccharide moiety is formed as a lipid-linked, membrane-associated glycan; (b) the lipid component in most cases is a polyisoprenoid phosphate; (c) the assembly of the lipid-linked saccharide intermediate depends on reactions taking place at both sides of the cell membrane, which requires the obligatory transmembrane movement of amphipathic molecules across the lipid bilayer. These general characteristics are present in the biosynthesis of the O-antigen component of the bacterial lipopolysaccharide, which serves as a model system to investigate the molecular and mechanistic basis of glycoconjugate synthesis, as summarized in this mini-review.

Structure-Guided Investigation of Lipopolysaccharide O-Antigen Chain Length Regulators Reveals Regions Critical for Modal Length Control

The O-antigen component of the lipopolysaccharide (LPS) represents a population of polysaccharide molecules with nonrandom (modal) chain length distribution. The number of the repeat O units in each individual O-antigen polymer depends on the Wzz chain length regulator, an inner membrane protein belonging to the polysaccharide copolymerase (PCP) family. Different Wzz proteins confer vastly different ranges of modal lengths (4 to > 100 repeat units), despite having remarkably conserved structural folds. The molecular mechanism responsible for the selective preference for a certain number of O units is unknown. Guided by the three-dimensional structures of PCPs, we constructed a panel of chimeric molecules containing parts of two closely related Wzz proteins from Salmonella enterica and Shigella flexneri which confer different O-antigen chain length distributions. Analysis of the O-antigen length distribution imparted by each chimera revealed the region spanning amino acids 67 to 95 (region 67 to 95), region 200 to 255, and region 269 to 274 as primarily affecting the length distribution. We also showed that there is no synergy between these regions. In particular, region 269 to 274 also influenced chain length distribution mediated by two distantly related PCPs, WzzB and FepE. Furthermore, from the 3 regions uncovered in this study, region 269 to 274 appeared to be critical for the stability of the oligomeric form of Wzz, as determined by cross-linking experiments. Together, our data suggest that chain length determination depends on regions that likely contribute to stabilize a supramolecular complex.

Membrane Topology and Identification of Critical Amino Acid Residues in the Wzx O-Antigen Translocase from Escherichia coli O157:H4

Wzx belongs to a family of membrane proteins involved in the translocation of isoprenoid lipid-linked glycans, which is loosely related to members of the major facilitator superfamily. Despite Wzx homologs performing a conserved function, it has been difficult to pinpoint specific motifs of functional significance in their amino acid sequences. Here, we elucidate the topology of the Escherichia coli O157 Wzx (Wzx(EcO157)) by a combination of bioinformatics and substituted cysteine scanning mutagenesis, as well as targeted deletion-fusions to green fluorescent protein and alkaline phosphatase. We conclude that Wzx(EcO157) consists of 12 transmembrane (TM) helices and six periplasmic and five cytosolic loops, with N and C termini facing the cytoplasm. Four TM helices (II, IV, X, and XI) contain polar residues (aspartic acid or lysine), and they may form part of a relatively hydrophilic core. Thirty-five amino acid replacements to alanine or serine were targeted to five native cysteines and most of the aspartic acid, arginine, and lysine residues. From these, only replacements of aspartic acid-85, aspartic acid-326, arginine-298, and lysine-419 resulted in a protein unable to support O-antigen production. Aspartic acid-85 and lysine-419 are located in TM helices II and XI, while arginine-298 and aspartic acid-326 are located in periplasmic and cytosolic loops 4, respectively. Further analysis revealed that the charge at these positions is required for Wzx function since conservative substitutions maintaining the same charge polarity resulted in a functional protein, whereas those reversing or eliminating polarity abolished function. We propose that the functional requirement of charged residues at both sides of the membrane and in two TM helices could be important to allow the passage of the Und-PP-linked saccharide substrate across the membrane.

Functional analysis of the C-terminal domain of the WbaP protein that mediates initiation of O antigen synthesis in Salmonella enterica

WbaP catalyzes the transfer of galactose-1-phosphate onto undecaprenyl phosphate (Und-P). The enzyme belongs to a large family of bacterial membrane proteins required for initiation of the synthesis of O antigen lipopolysaccharide and polysaccharide capsules. Previous work in our laboratory demonstrated that the last transmembrane helix and C-terminal tail region of WbaP (WbaP(CT)) are sufficient for enzymatic activity. Here, we demonstrate the cytoplasmic location of the WbaP C-terminal tail and show that WbaPCT domain N-terminally fused to thioredoxin (TrxA-WbaP(CT)) exhibits improved protein folding and enhanced transferase activity. Alanine replacement of highly conserved charged or polar amino acids identified seven critical residues for enzyme activity in vivo and in vitro. Four of these residues are located in regions predicted to be a-helical. These regions and their secondary structure predictions are conserved in distinct WbaP family members, suggesting they may contribute to form a conserved catalytic center.