Highly potent inhibitors of quorum sensing in Staphylococcus aureus revealed through a systematic synthetic study of the group-III autoinducing peptide.

Methods to intercept bacterial quorum sensing (QS) have attracted significant attention as potential anti-infective therapies. Staphylococcus aureus is a major human pathogen that utilizes autoinducing peptide (AIP) signals to mediate QS and thereby regulate virulence. S. aureus strains are categorized into four groups (I-IV) according to their AIP signal and cognate extracellular receptor, AgrC. Each group is associated with a certain disease profile, and S. aureus group-III strains are responsible for toxic shock syndrome and have been underestimated in other infections to date. A limited set of non-native AIP analogs have been shown to inhibit AgrC receptors; such compounds represent promising tools to study QS pathways in S. aureus . We seek to expand this set of chemical probes and report herein the first design, synthesis, and biological testing of AIP-III mimetics. A set of non-native peptides was identified that can inhibit all four of the AgrC receptors (I-IV) with picomolar IC50 values in reporter strains. These analogs also blocked hemolysis by wild-type S. aureus group I-IV strains-a virulence trait under the control of QS-at picomolar concentrations. Moreover, four of the lead AgrC inhibitors were capable of attenuating the production of toxic shock syndrome toxin-1 (also under the control of QS) by over 80% at nanomolar concentrations in a wild-type S. aureus group-III strain. These peptides represent, to our knowledge, the most potent synthetic inhibitors of QS in S. aureus known, and constitute new and readily accessible chemical tools for the study of the AgrC system and virulence in this deadly pathogen.

Role of intramolecular epitope spreading in pemphigus vulgaris.

Pemphigus vulgaris (PV) is an acquired immunobullous disorder. At the early stage of the disease (mucosal PV), patients display only autoimmunity to desmoglein (Dsg) 3 and develop mucosal blisters; while at the later stage of the disease (mucocutaneous PV), patients exhibit non-cross-reactive autoimmunity to both Dsg3 and Dsg1 and acquire cutaneous as well as mucosal blisters. At these two disease stages, Dsg3 autoantibodies exhibit different tissue-binding patterns and pathogenic activities, suggesting that they may recognize distinct epitopes. To test this hypothesis and to investigate the mechanism underlying the disease transition, we studied Dsg3 autoantibody epitopes from mucosal PV patients and patients exhibiting disease transition to mucocutaneous PV. We demonstrated that autoantibodies from the majority of mucosal PV patients target epitopes at the COOH-terminal portion of the Dsg3 ectodomain. Interestingly, only autoantibodies against the Dsg3 NH2-terminal epitope(s) are able to bind human skin. Moreover, we discovered that the intramolecular epitope spreading from Dsg3(87-566) to Dsg3(1-88) is a critical step that precedes the intermolecular epitope spreading from Dsg3 to Dsg1. During disease transition, this mechanism dictates the development of Dsg3 autoantibodies that recognize human skin and lead to expression of cutaneous PV lesions.

T cell receptor gene usage of BP180-specific T lymphocytes from patients with bullous pemphigoid and pemphigoid gestationis.

BP180 is the autoantigen of different immunobullous diseases, including bullous pemphigoid (BP) and pemphigoid gestationis (PG). Previously, we demonstrated that the NC16A domain of this autoantigen harbors key epitopes of autoantibodies and T cells, indicating that it plays an essential role in the pathogenesis of diseases. Moreover, NC16A-specific T cell clones derived from these patients were shown to express a CD4+ memory T cell phenotype and secrete cytokines that may promote autoantibody production. In this study, we further characterize the properties of these T cells by analyzing their epitope specificity and T cell receptor (TCR) gene usage. We discovered that 83% of T cell clones obtained from BP patients preferentially express TCRBV13, while clones derived from a PG patient express the TCRBV3 gene. However, no preferential TCRBJ gene usage was identified. In conclusion, our results provide an advanced understanding of the characteristics of autoimmune T cells in immunobullous diseases.

T cell receptor gene usage in desmoglein-3-specific T lymphocytes from patients with pemphigus vulgaris.

Pemphigus vulgaris (PV) is an autoimmune disease mediated by autoantibodies against desmoglein-3 (Dsg3). It has been documented that both humoral and cellular autoimmunity play essential roles in the development of PV. Recently, we identified that T cells from PV patients respond to three antigenic fragments on the ectodomain of Dsg3. These T cells are CD4 alpha/beta cells secreting a Th2-like cytokine profile, and responding of Dsg3 in a restriction to HLA-DRBI*0402 or 1401 alleles. Other characteristics of these cells, such as detailed epitope(s) and T cell receptors (TCRs) usage, however, have not been investigated. The purpose of this study is to determine detailed T cell epitope(s) and TCR genes utilized by Dsg3-specific T cells. Here, we found that Dsg3(AA145-192)-specific cells preferentially utilize the TCRVbeta13 gene, while Dsg3(AA240-303)- and Dsg3 (AA570-614)-specific cells utilize Vbeta7 and Vbeta17 genes, respectively. Analysis of TCRValpha gene expression, it appears that Valpha22 gene is expressed by Dsg3(AA145-192)-specific cells, whereas the Valpha10 gene is predominantly utilized by Dsg3(AA240-303)-specific T cells. There are no specific utilization of Valpha gene in the group of cells proliferate to Dsg3 (AA570-614). We believe that this information will further our understanding of the properties of autoimmune T cells in patients with PV.

Pathogenicity and epitope characteristics of anti-desmoglein-1 from pemphigus foliaceus patients expressing only IgG1 autoantibodies.

Pemphigus foliaceus (PF) is an antibody-mediated autoimmune disorder with IgG1 and IgG4 as the predominant subclasses of autoantibodies against a desmosomal glycoprotein, desmoglein-1 (Dsg1). Previously, we found that the IgG4 anti-Dsg1 autoantibodies only recognize a conformational epitope(s), whereas the IgG1 autoantibodies recognize both conformational and linear epitopes but do not display pathogenicity in the passive transfer animal model. The purpose of this study was to analyze the epitopes recognized by autoanti-bodies from a subset of PF patients who only express anti-Dsg1 of the IgG1 isotype throughout the course of their diseases and to further characterize the pathogenicity of their IgG1 anti-Dsg1. We found that IgG1 auto-antibodies in this subset of PF patients, similar to IgG4 autoantibodies from other PF patients, are able to bind both human and mouse skin and induce the experimental PF in mice. Moreover, a detailed epitope mapping reveals that the conformational epitopes recognized by IgG1 autoantibodies from these PF patients are restricted to the first 161 amino acids of Dsg1, whereas the linear epitopes are spread throughout the entire ectodomain. In conclusion, our study reveals that the isotype of IgG does not necessarily determine the epitopes and pathogenicity of pemphigus autoantibodies.